Search Results (644)

Figure 1
<p>Expression of CNN3 in mouse tissues and throughout myoblast differentiation. (<b>A</b>) Immunoblotting was conducted to assess CNN3 expression in C2C12 myoblasts and tissues from 8-week-old mice; α-tubulin was used as a loading control. (<b>B</b>) C2C12 myoblasts were collected and subjected to immunoblotting of MyoD, MyoG, MyHC, and CNN3 at designated differentiation time points; β-actin was used as a loading control. (<b>C</b>) Expression levels were normalized versus β-actin, and relative ratios were calculated, with day 0 set as the baseline for MyoD and CNN3, day 1 for MyoG, and day 2 for MyHC. Data are expressed as means ± SEM (<span class="html-italic">n</span> = 3), with asterisks denoting statistically significant differences (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 2
<p>CNN3 knockdown enhanced F-actin levels and increased nuclear YAP1. C2C12 myoblasts were transfected with 200 nM of either control scRNA or siCNN3 (siCNN3-1 or siCNN3-2). (<b>A</b>) CNN3 expressions were determined 24 h after transfection by immunoblotting. Expression levels were normalized versus β-actin, and relative ratios were calculated using the control scRNA as the baseline (set to one). (<b>B</b>) Cells were stained with FITC-phalloidin (green) to visualize F-actin and Hoechst 33,342 (blue) to label nucleus. Scale bar: 25 μm. Phalloidin fluorescence intensities were quantified using ImageJ program. (<b>C</b>) F-actin levels were analyzed by flow cytometry following staining with FITC-phalloidin. (<b>D</b>) Cytoplasmic and nuclear fractions were subjected to immunoblotting to detect YAP1, pYAP1 (phosphorylated YAP1), and CNN3. α-Tubulin and lamin B2 were used as markers for the cytoplasmic and nuclear fractions, respectively; β-actin was used as a loading control. (<b>E</b>) Expression levels were normalized versus β-actin, and relative ratios were calculated versus scRNA. Data are expressed as means ± SEM (<span class="html-italic">n</span> = 3), with asterisks denoting statistically significant differences (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 3
<p>CNN3 depletion facilitated myoblast proliferation. C2C12 myoblasts were transfected with either control scRNA or siCNN3 and analyzed 24 h post-transfection. (<b>A</b>) Cell proliferation was assessed using EdU incorporation (green) to label replicating cells, and Hoechst 33,342 (blue) was used to counterstain the nucleus. Scale bar: 50 µm. (<b>B</b>) The percentages of EdU-positive cells were determined using ImageJ program. (<b>C</b>) Viable cell numbers were measured using a cell viability assay kit. (<b>D</b>) mRNA levels of proliferation markers (PCNA, Cyclin B1, and Cyclin D1) were assessed by RT-<span class="html-italic">q</span>PCR and normalized versus GAPDH expression. (<b>E</b>,<b>F</b>) Cell cycle analysis was performed using flow cytometry with scatter plots. Data are expressed as means ± SEM (<span class="html-italic">n</span> = 3), with asterisks denoting statistically significant differences (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 4
<p>CNN3 knockdown suppressed expressions of myogenic factors. (<b>A</b>) C2C12 myoblasts were transfected with either control scRNA or siCNN3, allowed to differentiate, and subjected to immunoblotting at designated differentiation time points; β-actin was used as a loading control. Expression of myogenic regulatory factors and CNN3 were evaluated by immunoblotting. (<b>B</b>) Expression intensities of proteins in myoblasts transfected with scRNA (open column) and siCNN3 (blue column) were normalized versus β-actin. Results are expressed as relative ratios compared to scRNA levels on day 0 for CNN3 and MyoD and day 3 for MyoG and MyHC. Data are expressed as means ± SEM (<span class="html-italic">n</span> = 3), with asterisks denoting statistically significant differences (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 5
<p>Depletion of CNN3 disrupted myogenic differentiation. C2C12 myoblasts were transfected with control scRNA or siCNN3 and then allowed to differentiate for 5 days. (<b>A</b>) Representative immunocytochemistry results after staining with MyHC antibody (green) and Hoechst 33,342 (blue). Scale bar: 50 μm. (<b>B</b>) MyHC-positive areas, differentiation and fusion indices, and myotube widths were evaluated as outlined in <a href="#sec2dot7-cells-14-00142" class="html-sec">Section 2.7</a>. Data are expressed as means ± SEM (<span class="html-italic">n</span> = 3), with asterisks denoting statistically significant differences (*** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">

Figure 1
<p>Scheme of the new cell-stretching device in this study.</p> Full article ">Figure 2
<p>Device fabrication steps. (<b>A</b>) The upper and lower sheets were created from PDMS. (<b>B</b>) Device assembly and tubing process. The upper sheet and PDMS thin membrane were bonded using PDMS mortar, and the lower sheet and PDMS thin membrane were permanently bonded using plasma. (<b>C</b>) Photograph of the completed device. Scale bar = 10 mm.</p> Full article ">Figure 3
<p>Stretched PDMS thin membrane in the device. (<b>A</b>) Setup of the stretching apparatus and device. As indicated by the arrows, a PDMS membrane was drawn in the lower channel. (<b>B</b>) Cross-section of the device reconstructed from confocal microscope images. Scale bar = 300 µm. (<b>C</b>) Graph of PDMS thin membrane elongation at different vacuum pressures. Mean ± SD, <span class="html-italic">n</span> = 3.</p> Full article ">Figure 4
<p>Advantages of cell-stretching culture device with a detachable channel. (<b>A</b>) Schematic of microscopic observation with the upper sheet attached to the thin membrane. (<b>B</b>) Schematic of microscopic observation after removing both sheets from the thin membrane. (<b>C</b>) Preparation for microscopic observation. The upper sheet was removed from the PDMS thin membrane, and only the cell culture area of the thin membrane was cut out and placed on the coverslip. The membrane was cut in the number order shown in the figure. (<b>D</b>) Fluorescence microscopy image of a cell observed with 40× and 60× oil immersion objective lenses. Scale bar = 50 µm.</p> Full article ">Figure 5
<p>Effects of cyclic stretching on integrinβ1 and vinculin localization in OP9 cells. Fluorescence images of integrinβ1 (green), f-actin (red), and vinculin (gray) in OP9 cells cultured on the device after 72 h under static conditions followed by 24 h under (<b>A</b>) static or (<b>B</b>) cyclic stretching conditions (0.5 Hz) photographed with a 100× oil immersion objective lens. Confocal images were captured every 0.48 µm along the z-axis to create z-stacks (14 slices) for maximum-intensity projection (scanning zoom 2×). The right figure shows the fluorescence intensity–distance profiles of integrinβ1 (green), f-actin (red), vinculin (gray), and nuclei (blue) on lines (<b>a</b>–<b>d</b>) drawn on the microscope image (MIP).</p> Full article ">Figure 6
<p>Effects of cyclic stretching on YAP1 localization in OP9 cells. (<b>A</b>) Criteria for evaluating the subcellular localization of YAP1. Cross-sections through the center of the cell nucleus were selected, and profiles of nuclear and YAP1-derived fluorescence signal intensities at the cross-sections were obtained. Three patterns were observed: cells with YAP1 localized in the nucleus, cells with YAP1 localized in both the nucleus and cytoplasm, and cells with YAP1 localized in the cytoplasm. (<b>B</b>) Fluorescence images of OP9 cells in the static condition or in response to cyclic stretching (0.5 Hz) for 4 h after 72 h of static culture. Confocal images were acquired every 0.51 µm along the z-axis to create z-stacks (35 slices) for maximum-intensity projection. Scale bar = 30 µm. (<b>C</b>) The percentage of cells with YAP localization in the nucleus under the culture conditions is shown in (<b>B</b>), with an average of four microscopy images taken from four different devices. N = nucleus. C = cytoplasm. <span class="html-italic">p</span> = 0.013, * <span class="html-italic">p</span> &gt; 0.05.</p> Full article ">

Figure 1
<p>Identification of cell polarity proteins interacting with YAP and TAZ in liver cancer cells. Volcano plot showing a significant binding of proteins to YAP (<b>A</b>) or TAZ (<b>B</b>) identified by LC-MS analysis. Proteins known to contribute to cell polarity are indicated. The vertical line indicates a two-fold enrichment for binding interactions. The horizontal line represents a false discovery rate (FDR) of <span class="html-italic">p</span> ≤ 0.05.</p> Full article ">Figure 2
<p>Functional screen for cell polarity factors that regulate YAP and TAZ. (<b>A</b>) Table summarizing proteins contributing to cell polarity (e.g., apical, lateral, and basolateral complex constituents). Two siRNAs for each gene were transfected in HepG2 cells, and endogenous YAP or TAZ localization was investigated utilizing immunofluorescence. (+) = more than 1/3 of cells show a strong nuclear YAP/TAZ enrichment with both siRNAs; (-) = less than 1/3 of cells or weak nuclear enrichment was observed. (<b>B</b>) Exemplary pictures of immunofluorescent YAP and TAZ stains are shown after the transfection of scrambled siRNA (scr. siRNA, negative control), Large tumor suppressor kinase 1/2 siRNAs (positive control), MPP5 siRNAs and AMOTL2 siRNAs for 48 h. Bars: 25 µm.</p> Full article ">Figure 3
<p>MPP5 physically interacts with YAP/TAZ. (<b>A</b>) Co-IP with endogenous YAP, TAZ, and MPP5 proteins in HepG2 cells. (<b>B</b>) Co-IP after overexpression of YAP or TAZ (upper and lower blot, respectively) for 48 h in HepG2 cells. (<b>C</b>) Scheme of human MPP5 and mutant isoforms used for Co-IP experiments. (<b>D</b>) Western immunoblot analysis confirmed the efficient expression of all MPP5 isoforms. The control shows the endogenous MPP5 expression (ctrl.). (<b>E</b>) Co-IP experiment after overexpression of HA-tagged YAP and all MPP5 isoforms for 48 h. MPP5 is detected after the pull-down of YAP using an HA-specific antibody. The arrow indicates the position where the MPP5 isoform was expected. Total protein lysate was used as input control for (<b>A</b>,<b>B</b>,<b>E</b>). Rabbit serum IgG served as negative control (IgG ctrl./antibody ctrl.).</p> Full article ">Figure 4
<p>MPP5 is a negative regulator of YAP activity. (<b>A</b>) IF microscopy detecting endogenous MPP5 in HepG2, HuH6, and HLF cells. The cell density was chosen to create both dense and subconfluent areas. Scale bars: 25 µm. (<b>B</b>) PLA for YAP and MPP5 in different liver cancer cell lines. MPP5 and YAP alone represent negative controls, while the combined administration illustrates their cytoplasmic and partly membranous co-localization. (<b>C</b>) Western immunoblot of total protein fractions after MPP5 inhibition using two siRNAs (#1 and #2). (<b>D</b>) Western immunoblot of cytoplasmic and nuclear protein fractions after MPP5 inhibition. Poly (ADP-ribose) polymerase (PARP) and tubulin were fractionated controls. (<b>E</b>) Heatmap illustrating the expression of YAP target genes (Wang signature, <span class="html-italic">n</span> = 22) after inhibition of MPP5. The efficient knockdown of <span class="html-italic">MPP5</span> is illustrated in the first line. The lower section of the heatmap displays a balanced score for two YAP-dependent gene signatures (Wang signature and Cordenonsi signature, <span class="html-italic">n</span> = 57). For (<b>C</b>–<b>E</b>), untreated cells (ctrl.) and scrambled siRNA (scr.)-transfected cells were used as controls. For Western blot quantification, pYAP and YAP signals were measured with the Fiji software (ImageJ2 Version 2.14.0) and normalized to endogenous YAP (<b>C</b>) or nuclear PARP (<b>D</b>). The results of signal quantification are shown under the respective protein panels.</p> Full article ">Figure 5
<p>Membranous MPP5 expression negatively correlates with nuclear YAP/TAZ enrichment in HCC cells. (<b>A</b>) Immunohistochemical analysis of MPP5, YAP, TAZ, MCM2, and Ki-67. Higher magnifications are shown in the upper right corner. Scale bars: 50 µm. (<b>B</b>) Proportional bar charts illustrate the correlation of IHC stains with tumor grading. IHC scores (white: low score, grey: medium score, black: high score) were correlated with tumor grading (0: normal liver tissue, 1: G1, 2: G2, 3: G3, 4: G4). Spearman’s correlation was used for statistical testing.</p> Full article ">Figure 6
<p><span class="html-italic">MPP5</span> negatively correlates with YAP target gene expression and clinical outcome in HCC patients. (<b>A</b>) Transcriptome analysis of <span class="html-italic">MPP5</span> mRNA levels in HCC tissues compared to surrounding liver tissues. Mann–Whitney U test (** <span class="html-italic">p</span> ≤ 0.01). (<b>B</b>) Distribution of MPP5 expression in HCCs. Tumor tissues with MPP5 levels lower than 75% of non-malignant tissues were classified as ‘reduced’. (<b>C</b>) Association between MPP5 and YAP target gene signature expression (Wang and Cordenonsi signatures). Spearman correlation analysis. (<b>D</b>) Higher MPP5 transcript levels (red) significantly correlate with better overall survival and disease-free survival of HCC patients. Respective Kaplan–Meier curves are shown.</p> Full article ">

Figure 1
<p>Modular structure of YAP.</p> Full article ">Figure 2
<p>Mechano-transduction pathways regulating YAP/TAZ activity. This schematic shows how mechanical signals—such as ECM stiffness, mechanical stretch, and shear stress—modulate YAP/TAZ activity via Hippo-dependent and independent pathways. The Hippo pathway’s core kinases, MST1/2 and LATS1/2, respond to mechanical cues to regulate YAP/TAZ phosphorylation and localization. Alternatively, these mechanical stimuli can bypass the kinase cascade, influencing YAP/TAZ localization through cytoplasmic and nuclear actin dynamics.</p> Full article ">Figure 3
<p>Hippo/YAP signaling pathway in physical and rehabilitation medicine. The Hippo/YAP pathway responds to mechanical stimuli, promoting tissue repair and regeneration in cartilage, bone, muscle, blood vessels, nerves, fascia, and tendons. YAP activation under different forms of mechanical loading—such as exercise, LIPUS, vibration therapy, etc.—reduces inflammation and supports cell proliferation and differentiation, as well as the regeneration and remodeling of various tissues. This pathway modulates key cellular processes, making it essential for tailoring rehabilitation protocols to enhance recovery in musculoskeletal injuries and degenerative conditions.</p> Full article ">

Figure 1
<p>(<b>a</b>) Structure of PARP1 in rainbow scale colors, ID: 4DQY and (<b>b</b>) A Ramachandran diagram analysis of PARP-1 after a 500 ns simulation in a rainbow scale: red indicates more accumulation, and blue indicates less accumulation of residuals.</p> Full article ">Figure 2
<p>PARP-1 stabilization properties after a 500 ns molecular dynamics simulation: (<b>a</b>) RMSD; (<b>b</b>) RMSF; (<b>c</b>) Radius of gyration; (<b>d</b>) Number of hydrogen bonds.</p> Full article ">Figure 3
<p>(<b>a</b>) Structure of YAP-1 in rainbow scale colors, ID: 6GEI and (<b>b</b>) A Ramachandran diagram analysis of YAP-1 after a 500 ns simulation in a rainbow scale: red indicates more accumulation, and blue indicates less accumulation of residuals.</p> Full article ">Figure 4
<p>Stabilization properties of YAP-1 after a 500 ns molecular dynamics simulation: (<b>a</b>) RMSD; (<b>b</b>) RMSF; (<b>c</b>) Radius of gyration; (<b>d</b>) Number of hydrogen bonds.</p> Full article ">Figure 5
<p>Two-dimensional structure of naturally occurring and synthetic PARP-1 inhibitors.</p> Full article ">Figure 6
<p>Two-dimensional structure of naturally occurring and synthetic YAP-1 inhibitors.</p> Full article ">Figure 7
<p>Druggable pockets of (<b>a</b>) PARP-1 y (<b>b</b>) YAP-1.</p> Full article ">Figure 8
<p>PARP-1 stabilization properties with inhibitors after a 100 ns molecular dynamics simulation: (<b>a</b>) RMSD; (<b>b</b>) RMSF; (<b>c</b>) Radius of gyration; (<b>d</b>) Number of hydrogen bonds.</p> Full article ">Figure 9
<p>Stabilization properties of YAP-1 with the inhibitors after a 100 ns molecular dynamics simulation: (<b>a</b>) RMSD; (<b>b</b>) RMSF; (<b>c</b>) Radius of gyration; (<b>d</b>) Number of hydrogen bonds.</p> Full article ">Figure 10
<p>Structure of PARP-1 interacting with molecules: (<b>a</b>) ODK and (<b>b</b>) DK5.</p> Full article ">Figure 11
<p>Structure of YAP-1 interacting with the molecules (<b>a</b>) VER (<b>b</b>) GK4.</p> Full article ">Figure 11 Cont.
<p>Structure of YAP-1 interacting with the molecules (<b>a</b>) VER (<b>b</b>) GK4.</p> Full article ">Figure 12
<p>Electrostatic potential of (<b>a</b>) ODK-PARP−1, (<b>b</b>) DK5-PARP−1, (<b>c</b>) VER-YAP1, (<b>d</b>) GK4-YAP−1. Central image represents the overall electrostatic potential map.</p> Full article ">Figure 13
<p>Ramachandran diagram analysis of (<b>a</b>) ODK and (<b>b</b>) DK5 in a rainbow scale: red indicates more accumulation, and blue indicates less accumulation of residuals.</p> Full article ">Figure 14
<p>Ramachandran diagram analysis of (<b>a</b>) VER and (<b>b</b>) GK4 in a rainbow scale: red indicates more accumulation, and blue indicates less accumulation of residuals.</p> Full article ">

Figure 1
<p>Common transcriptional regulation between hepatoblastoma and medulloblastoma tumors: cross-combat normalization between GSE37418, GSE44971, and GSE104766 datasets. (<b>A</b>) Principal component analysis (PCA) performed on whole transcriptome before and after combat batch correction (HB: hepatoblastoma and MB: medulloblastoma) and control samples (normal cerebellum and normal liver). (<b>B</b>) Unsupervised clustering (Euclidean distances) based on the common HB-MB signature (1259 DEGs). (<b>C</b>) Volcano plot highlighting the significant transcription factors (light blue and annotated) regulated between tumors (HB and MB) and normal tissues (cerebellum and liver).</p> Full article ">Figure 2
<p>Embryonic program of LEF1 genome binding occupancy after WNT3A stimulation: genomic heatmap illustrating the enrichment of genomic intervals (hg38) for LEF1 binding in H1 human embryonic stem cell chromatin after WNT3A stimulation. From left to right, respective enrichment was shown for ChIP-seq of H1 input chromatin, LEF1 in unstimulated H1 cells, H3 histone lysine 27 trimethylation (repressive mark), H3 histone lysine 27 acetylation (active mark), and LEF1 in WNT3A-stimulated H1 cells. (TSS: transcription start site and TTS: transcription termination site).</p> Full article ">Figure 3
<p>LEF1 embryonic binding program predicts the WNT subtype in the medulloblastoma transcriptome: integration of the LEF1 binding program in the MB-PBTA transcriptome cohort. (<b>A</b>) Expression of LEF1 in the medulloblastoma transcriptome of the PBTA cohort stratified by medulloblastoma subtypes: WNT, SHH, G3, and G4. (<b>B</b>) Volcano plot of differentially expressed genes (with embryonic LEF1 binding) between the WNT-MB subtype and other MB samples. (<b>C</b>) Principal component analysis (PCA) performed on the 141 LEF1 embryonic targets overexpressed in the WNT-MB subtype. (<b>D</b>) Unsupervised clustering (Euclidean distances) based on the expression of the 141 LEF1 embryonic targets overexpressed in the WNT-MB subtype.</p> Full article ">Figure 4
<p>Heterogeneity of LEF1-dependent embryonic promoters for targets activated during WNT medulloblastoma: integration into the MB-PBTA cohort. (<b>A</b>) Scatterplot of the integrative analysis representing ChIP-seq peak distances from TSS versus log2 fold changes in the WNT-MB transcriptome. Dot sizes represent peak scores from the MACS2 algorithm for LEF1-H1-WNT3A ChIP-seq data. (<b>B</b>) Promoter visualization for the LEF1 and LEF1-AS loci. (<b>C</b>) Promoter visualization for the DKK4 locus. (<b>D</b>) Promoter visualization for the RNF43 locus. (<b>E</b>) Promoter visualization for the MYCN locus.</p> Full article ">Figure 5
<p>WNT signaling LEF1-dependent signature in medulloblastoma: MB-PBTA cohort. (<b>A</b>) Functional enrichment network based on LEF1 targets activated during the WNT-medulloblastoma subtype using the Gene Ontology Biological Process (GO-BP) database. (<b>B</b>) Unsupervised clustering (Euclidean distances) of the WNT-program LEF1-dependent genes in medulloblastoma tumors. (<b>C</b>) Principal component analysis (PCA) of the WNT-program LEF1-dependent genes in medulloblastoma tumors. (<b>D</b>) Univariate binomial regression analysis of the 13 WNT signaling target genes of LEF1 during medulloblastoma (outcome: WNT subtype).</p> Full article ">Figure 6
<p>Validation of the WNT-LEF1 signature in independent medulloblastoma cohorts: GSE37418 and scRNA-seq GSE155446 datasets. (<b>A</b>) Unsupervised clustering (Euclidean distances) based on the expression of the WNT-LEF1-dependent program (GSE37418). (<b>B</b>) Principal component analysis (PCA) of the WNT-LEF1-dependent program (GSE37418). (<b>C</b>) ElasticNet tuning of lambda and alpha parameters to predict the WNT subgroup using the WNT-LEF1-dependent program. (<b>D</b>) Barplot of ElasticNet coefficients predicting the WNT subtype in the GSE37418 cohort. (<b>E</b>) WNT-MB subtypes in the single-cell transcriptome dataset GSE155446 (28 MB tumor samples). (<b>F</b>) WNT-LEF1-dependent score in the single-cell RNA-seq dataset GSE155446.</p> Full article ">Figure 7
<p>Validation of the WNT-LEF1 signature in an independent hepatoblastoma cohort: GSE131329 and scRNA-seq GSE180655 datasets. (<b>A</b>) Unsupervised clustering (Euclidean distances) based on the expression of the WNT-LEF1 program (GSE131329). (<b>B</b>) Principal component analysis (PCA) of the WNT-LEF1 program (GSE131329). (<b>C</b>) UMAP dimensionality reduction showing sample identities in the scRNA-seq dataset GSE180655. (<b>D</b>) UMAP dimensionality reduction showing cell-type identities in the scRNA-seq dataset GSE180655. (<b>E</b>) WNT-LEF1-dependent score in the single-cell RNA-seq dataset GSE180655, stratified by sample types (background: adjacent normal liver; pdx: patient-derived xenotransplantation model; tumor: HB tumor samples). (<b>F</b>) WNT-LEF1-dependent score in the single-cell RNA-seq dataset GSE180655, stratified by cell types.</p> Full article ">

Figure 1
<p>(<b>a</b>) Map of Micronesia in relation to Papua New Guinea, showing locations of Yap, Palau, Guam and Pohnpei. (<b>b</b>) Map of Yap (Wa’ab) islands showing location of coral reef samples described in <a href="#diversity-17-00034-t001" class="html-table">Table 1</a> (large, numbered dots; small dots are locations of freshwater samples in 1988). Scale = 5 km.</p> Full article ">Figure 2
<p><span class="html-italic">Podosira</span> and <span class="html-italic">Paralia.</span> (<b>a</b>) <span class="html-italic">Podosira hormoides</span>, valve view, LM. (<b>b</b>,<b>c</b>) <span class="html-italic">Podosira montagnei</span> in valve and girdle views, LM. (<b>d</b>–<b>g</b>) <span class="html-italic">Paralia longispina.</span> (<b>d</b>,<b>e</b>) Frustules in valve and girdle views, LM. (<b>f</b>,<b>g</b>) SEM images of frustules. (<b>f</b>) Separating valve and girdle bands in girdle view. (<b>g</b>) Separating valve and linking valve, the latter showing interior aspect with internal striae. Scale bars: (<b>a</b>–<b>e</b>) = 10 µm, (<b>f</b>,<b>g</b>) = 5 µm.</p> Full article ">Figure 3
<p><span class="html-italic">Ehrenbergiopsis</span> and <span class="html-italic">Actinocyclus</span>. (<b>a</b>–<b>d</b>) <span class="html-italic">Ehrenbergiopsis hauckii</span>. (<b>a</b>) LM. (<b>b</b>) SEM external of papillate valve. (<b>c</b>,<b>d</b>) SEM of internal valve faces showing absence of fultoportulae and rimoportulae. (<b>e</b>–<b>g</b>) <span class="html-italic">Actinocyclus decussatus</span>. (<b>e</b>) Series of focal planes from low to high of a slightly tilted valve. Arrow points to pseudonodulus, arrowhead to one of the many rimoportulae. (<b>f</b>) Interior aspect in SEM showing pseudonodulus (arrow) and rimoportulae (arrowhead). (<b>g</b>) Frustule in oblique view showing valve contours, SEM. Scale bars: (<b>a</b>–<b>c</b>,<b>e</b>–<b>g</b>) = 10 µm, (<b>d</b>) = 2 µm.</p> Full article ">Figure 4
<p><span class="html-italic">Actinocyclus</span> and <span class="html-italic">Roperia</span>. (<b>a</b>–<b>c</b>) <span class="html-italic">Actinocyclus subtilis</span>. (<b>a</b>) Whole valve, LM. (<b>b</b>,<b>c</b>) Portions of valves to show areolae without clear interfascicular rows and central area with numerous areolae separated by a hyaline ring, LM and SEM (internal), respectively. (<b>d</b>–<b>h</b>) <span class="html-italic">Roperia tesselata</span>. (<b>d</b>,<b>e</b>) Valves with decussate areola pattern in LM and SEM (external). (<b>f</b>) Detail of cribrate areolae in external view, SEM. (<b>g</b>) Valve with less regular decussate pattern, external SEM. (<b>h</b>) Internal aspect, SEM, showing rimoportulae. Scale bars: (<b>a</b>) = 25 µm, (<b>b</b>–<b>e</b>,<b>g</b>,<b>h</b>) = 10 µm, (<b>f</b>) = 5 µm.</p> Full article ">Figure 5
<p>(<b>a</b>) <span class="html-italic">Asterolampra marylandica</span>, valve in LM. (<b>b</b>,<b>c</b>) <span class="html-italic">Proboscia alata</span> valve and detail of tip, SEM. (<b>d</b>–<b>h</b>) <span class="html-italic">Rhizosolenia imbricata</span>. (<b>d</b>)Valve and several girdle bands in dorsiventral view, LM. (<b>e</b>) Girdle bands in lateral view, SEM. (<b>f</b>) Long fragment of frustule showing arrangement of girdle bands in lateral rows, SEM. (<b>g</b>,<b>h</b>) Details of valve (V) and attached girdle bands in lateral and slightly oblique view, SEM, showing the spine on one side (arrow in <a href="#diversity-17-00034-f005" class="html-fig">Figure 5</a>g; out of view to left in <a href="#diversity-17-00034-f005" class="html-fig">Figure 5</a>h) and the matching groove (arrow, <a href="#diversity-17-00034-f005" class="html-fig">Figure 5</a>h), extending onto the girdle. Scale bars (<b>f</b>) = 25 µm, (<b>a</b>,<b>b</b>,<b>d</b>,<b>f</b>,<b>g</b>) = 10 µm, (<b>c</b>,<b>e</b>) = 5 µm.</p> Full article ">Figure 6
<p>(<b>a</b>–<b>c</b>). <span class="html-italic">Hemiaulus sinensis</span> valves in girdle view in LM and SEM. (<b>d</b>,<b>e</b>) <span class="html-italic">Anaulus minutus</span>, SEM. (<b>f</b>) <span class="html-italic">Bacteriastrum furcatum</span>, LM. Scale bars: (<b>a</b>,<b>b</b>,<b>d</b>,<b>f</b>) = 10 µm, Figures (<b>c</b>,<b>e</b>) = 5 µm.</p> Full article ">Figure 7
<p>(<b>a</b>–<b>d</b>) <span class="html-italic">Chaetoceros peruvianus</span>. (<b>a</b>) Upper valve in LM, showing base of setae. (<b>b</b>) Upper valve in SEM, in girdle view, showing large external rimoportula tube (arrow) between recurved setae. (<b>c</b>,<b>d</b>) Lower valve with straight setae and detail of pores and spines on seta. (<b>e</b>) <span class="html-italic">Disymmetria excentrica</span>, SEM. Scale bars: (<b>a</b>–<b>c</b>) = 10 µm, (<b>e</b>) = 5 µm, (<b>d</b>) = 2 µm.</p> Full article ">Figure 8
<p>(<b>a</b>–<b>c</b>) <span class="html-italic">Skeletonema grevillei</span>, SEM. (<b>a</b>) Portion of chain. (<b>b</b>) Two cells in chain connected by linked extensions of the fultoportulae, joined in the middle. (<b>c</b>) Oblique view of internal surface (center of valve missing), showing openings of the single rimoportula (arrow) and the ring of fultoportulae (arrowheads). (<b>d</b>–<b>f</b>) Unidentified Cymatosiraceae in LM and two views of a valve in SEM, (<b>f</b>) rotated and tilted 60°. Scale bars: (<b>a</b>) = 25 µm, (<b>d</b>) = 10 µm, (<b>e</b>,<b>f</b>) = 5 µm, (<b>b</b>,<b>c</b>) = 2 µm.</p> Full article ">Figure 9
<p>(<b>a</b>,<b>b</b>). <span class="html-italic">Odontella obtusa</span>, SEM. (<b>c</b>,<b>d</b>) <span class="html-italic">Pseudictyota dubia</span>. (<b>c</b>) Valve in valve view, LM. (<b>d</b>) Valve in girdle view, SEM, showing ocelli (arrowhead), rimoportulae (arrow) and pseudoloculate structure. Scale bars: (<b>a</b>) = 25 µm, (<b>b</b>,<b>c</b>) = 10 µm, (<b>d</b>) = 5 µm.</p> Full article ">Figure 10
<p>(<b>a</b>–<b>f</b>) <span class="html-italic">Ardissoneopsis</span>. (<b>a</b>,<b>b</b>) <span class="html-italic">A. gracilis</span> valve from Y36-2 in LM with central portion and one pole; detail of central portion, typical of the species. (<b>c</b>–<b>f</b>) Valve fragments from Y26C, possibly a different species but not <span class="html-italic">A. undosa</span>. (<b>c</b>) Mid portion with inflation, LM. (<b>d</b>) Internal SEM of pole, showing increased stria density at apex. (<b>e</b>) External view of apex, SEM, showing spines; broken edge shows lack of internal costae. (<b>f</b>) Internal aspect of central portion, SEM, showing location of annulus (arrow). Scale bars: (<b>a</b>) = 25 µm, (<b>b</b>–<b>f</b>) = 10 µm.</p> Full article ">Figure 11
<p>(<b>a</b>) <span class="html-italic">Climacosphenia elegantissima</span>, apical portion of valve and valvocopula showing parallel sides and annular lines until the larger space between craticular bars, LM. (<b>b</b>) <span class="html-italic">Climacosphenia scimiter</span>, valvocopula, LM. (<b>c</b>) <span class="html-italic">Grunowago pacifica</span>, SEM of valve interior and valvocopula, showing central costa. (<b>d</b>,<b>e</b>) <span class="html-italic">Synedrosphenia gomphonema</span>, LM. (<b>f</b>) <span class="html-italic">Synedrosphenia licmophoropsis</span>, apical and middle portions in LM, arrows indicate annulus. Scale bars: (<b>a</b>,<b>b</b>,<b>d</b>) = 25 µm, (<b>c</b>) = 20 µm, (<b>e</b>,<b>f</b>) = 10 µm.</p> Full article ">Figure 12
<p>(<b>a</b>) <span class="html-italic">Toxarium hennedyanum</span>, central portion showing smooth valve outline and field of scattered areolae inside the annulus; LM. (<b>b</b>) <span class="html-italic">Toxarium</span> cf. <span class="html-italic">hennedyanum</span> central and apical portions of a valve with no areolae inside the annulus (annulus along the valve margin with a line of areolae on each side); LM. (<b>c</b>) <span class="html-italic">Toxarium undulatum</span>, center and apical portions of valves showing undulating outline and scattered areolae inside the annulus at center and pole; SEM. Scale bars = 10 µm.</p> Full article ">Figure 13
<p>(<b>a</b>) <span class="html-italic">Biddulpha biddulphiana</span>, oblique valve in LM. (<b>b</b>–<b>d</b>) <span class="html-italic">Biddulphiella tridens</span>, SEM. (<b>b</b>) Exterior view showing deep sulci, spines, and two rimoportula tubules (arrows). (<b>c</b>) Valve in profile, tilt = 80°. (<b>d</b>) Valve as in (c), tilt = 0°, inset showing internal rimoportula opening. (<b>e</b>–<b>h</b>) <span class="html-italic">Biddulphiella cuniculopsis</span>, n. sp. (<b>e</b>) LM holotype valve from Guam at two focal planes in near-apical girdle view, showing rimoportula (arrow). (<b>f</b>) LM specimen from Yap, showing sulcus (arrowhead). (<b>g</b>,<b>h</b>) SEM of valve from Yap. (<b>g</b>) Interior, tilt = 40°, showing sulci (arrowheads) and possible rimoportula opening (arrows; compare <a href="#diversity-17-00034-f013" class="html-fig">Figure 13</a>e). (<b>h</b>) Valve in lateral girdle view, showing typical shape; arrowhead = sulcus. Scale bars: (<b>a</b>–<b>g</b>) = 10 µm, (<b>h</b>) = 5 µm.</p> Full article ">Figure 14
<p>(<b>a</b>–<b>c</b>) <span class="html-italic">Biddulphiopsis membranacea</span>, LM. (<b>a</b>,<b>b</b>). Complete valve (edges indicated by arrowheads) and detail of center showing scattered pattern contrasting with radiating striae. (<b>c</b>) Copula with apical septa. (<b>d</b>,<b>e</b>) <span class="html-italic">Lampriscus shadboltianus</span>. Valves in valve and girdle view, respectively, SEM, showing the smooth outline of the nominate variety and the characteristic spines on the edges of the ocelli in this species (arrow). Scale bars: (<b>a</b>,<b>c</b>) = 25 µm, (<b>b</b>,<b>d</b>,<b>e</b>) = 10 µm.</p> Full article ">Figure 15
<p>(<b>a</b>) <span class="html-italic">Striatella unipunctata</span>, SEM. (<b>b</b>,<b>c</b>) <span class="html-italic">Neofragilaria anomala</span>, internal views of two valves. (<b>d</b>–<b>h</b>). <span class="html-italic">Plagiogramma porcipellis</span>. (<b>d</b>) Two frustules in girdle view, LM. (<b>e</b>) Valve in LM. (<b>f</b>) Frustule in oblique view, SEM, showing spines, apical pores fields, central elevation, and broad valvocopula. (<b>g</b>) Detail of apical pore field and areolae, SEM. (<b>h</b>) Internal view of valve showing pseudoseptum and transverse costae. Scale bars: (<b>a</b>,<b>d</b>,<b>e</b>,<b>f</b>,<b>h</b>) = 10 µm, (<b>b</b>,<b>g</b>) = 5 µm. (<b>c</b>) = 2 µm.</p> Full article ">Figure 16
<p>(<b>a</b>,<b>b</b>) <span class="html-italic">Psammodiscus nitidus</span> in LM and SEM external view. (<b>c</b>) <span class="html-italic">Rhaphoneis castracanei</span>, LM. (<b>d</b>) <span class="html-italic">Perissonoë crucifera</span>, SEM external view. (<b>e</b>) <span class="html-italic">Bleakeleya notata</span>, LM. (<b>f</b>) <span class="html-italic">Perideraion montgomeryi</span>, SEM, external. (<b>g</b>,<b>h</b>) <span class="html-italic">Falcula paracelsiana</span>, SEM external, detail of apex with slits. Scale bars: (<b>h</b>) = 25 µm, (<b>a</b>,<b>c</b>–<b>e</b>) = 10 µm, (<b>b</b>,<b>f</b>,<b>g</b>) = 5 µm.</p> Full article ">Figure 17
<p><span class="html-italic">Hendeyella.</span> (<b>a</b>,<b>b</b>) <span class="html-italic">Hendeyella lineata</span> in SEM. (<b>a</b>) Yap voucher (Y37-8). (<b>b</b>) Guam specimen (GU44BF-1A) showing broad spines (arrowhead), broad valvocopula (VC), ligulate copula (arrow) and apical pore fields. (<b>c</b>–<b>f</b>) <span class="html-italic">Hendeyella rhombica</span>. (<b>c</b>) Chain in girdle view with valve view, LM. (<b>d</b>) Chain in girdle view showing broad valvocopula (VC) and narrowly branched spines, SEM. (<b>e</b>,<b>f</b>) Valve interiors, SEM, the latter showing weak heteropolarity, SEM. Scale bars: (<b>a</b>,<b>c</b>) = 10 µm, (<b>b</b>,<b>d</b>–<b>f</b>) = 5 µm.</p> Full article ">Figure 18
<p><span class="html-italic">Hyalosynedra</span> vs. <span class="html-italic">Stricosus.</span> (<b>a</b>–<b>f</b>) <span class="html-italic">Hyalosynedra laevigata</span>. (<b>a</b>) Valve in LM (Y41-7). (<b>b</b>) Fractured frustule in SEM (Y41-7), left-hand apex out of frame, shown in <a href="#diversity-17-00034-f018" class="html-fig">Figure 18</a>d. (<b>c</b>,<b>d</b>) Internal view of valve fragment (Y45-5) and detail of apex showing asymmetrical rimoportula (arrow). (<b>e</b>) Detail of apex external showing shallow ocellulimbus (arrow) and apical spines. (<b>f</b>,<b>g</b>) <span class="html-italic">Stricosus cardinalii</span> apex internal detail showing symmetrical rimoportula (arrow), and whole of same valve showing similarity of size and shape to <span class="html-italic">H. laevigata</span>. (<b>h</b>,<b>i</b>) <span class="html-italic">Stricosus harrisonii</span>, LM. Scale bars: (<b>h</b>) = 25 µm, (<b>a</b>–<b>c</b>,<b>g</b>,<b>i</b>) = 10 µm, (<b>d</b>–<b>f</b>) = 2 µm.</p> Full article ">Figure 19
<p>(<b>a</b>,<b>b</b>) <span class="html-italic">Neosynedra provincialis</span> valve and detail of apex, SEM. (<b>c</b>,<b>d</b>). <span class="html-italic">Neosynedra tortosa</span> apex and valve, SEM. (<b>e</b>–<b>h</b>). <span class="html-italic">Opephora pacifica</span>. (<b>e</b>) Frustule in LM. (<b>f</b>,<b>g</b>) Valve interiors in SEM, showing size range and heteropolarity. (<b>h</b>) Apex external view showing pore field, SEM. (<b>i</b>,<b>j</b>) <span class="html-italic">Synedra lata</span>, SEM. Scale bars: (<b>a</b>,<b>d</b>–<b>f</b>,<b>i</b>,<b>j</b>) = 10 µm, (<b>b</b>,<b>c</b>) = 5 µm, (<b>h</b>) = 2 µm.</p> Full article ">Figure 20
<p>(<b>a</b>–<b>d</b>) <span class="html-italic">Tabularia parva</span>, SEM. (<b>a</b>,<b>b</b>) Large specimen and detail of striae. (<b>c</b>,<b>d</b>) Small specimens, (<b>c</b>) oblique, showing lack of break in striae at valve–mantle junction. (<b>e</b>) <span class="html-italic">Grammatophora angulosa</span>, girdle view showing characteristically hooked septa, LM. (<b>f</b>,<b>g</b>) <span class="html-italic">Grammatophora oceanica</span>, LM, valve view and girdle view. (<b>h</b>) <span class="html-italic">Hyalosira tropicalis</span>, SEM. (<b>i</b>) <span class="html-italic">Microtabella interrupta</span>, SEM of frustule showing valve interior and copulae with septa. Scale bars: (<b>a</b>,<b>e</b>–<b>g</b>,<b>i</b>) = 10 µm, (<b>c</b>,<b>h</b>) = 5 µm, (<b>b</b>,<b>d</b>) = 2 µm.</p> Full article ">Figure 21
<p>(<b>a</b>) <span class="html-italic">Microtabella interrupta</span> valve and girdle bands in LM. (<b>b</b>,<b>c</b>) <span class="html-italic">Microtabella rhombica.</span> (<b>b</b>) SEM, arrow points to apical spine. (<b>c</b>) Valve and girdle band in LM. (<b>d</b>) <span class="html-italic">Cyclophora minor</span> frustule in LM, one valve with pseudoseptum. (<b>e</b>) <span class="html-italic">Cyclophora tenuis</span> valve exterior in SEM, arrows indicate two parts of the apical slit field. (<b>f</b>) <span class="html-italic">Licmophora flabellata</span> valve in LM, showing multiple rimoportulae along sternum. Scale bars: (<b>a</b>–<b>c</b>,<b>e</b>,<b>f</b>) = 10 µm, (<b>d</b>) = 2 µm.</p> Full article ">Figure 22
<p><span class="html-italic">Licmophora.</span> (<b>a</b>–<b>d</b>) <span class="html-italic">L. hastata</span>. (<b>a</b>,<b>b</b>) Valve interior with some of the girdle bands, SEM: valvocopula (VC), 1st pleura (1) and 2nd pleura (2); arrowhead on (<b>a</b>) points to thickened ligule on 2nd pleura, arrow to the rimoportula. (<b>c</b>) Valve with valvocopula (showing window in septum—arrowhead) in LM. (<b>d</b>) External SEM of valve with basal rimoportula opening (arrowhead), the five slits of the multiscissura below it, also showing apical pine (arrow). (<b>e</b>,<b>f</b>) <span class="html-italic">L. johnwestii</span>. (<b>e</b>) Interior view of valve with valvocopula showing the bridge-like septum (arrow) and the change in stria density between base and apex. (<b>f</b>) Partial frustule in girdle view, the valve across the bottom of image, showing valvocopula (VC), 1st pleura (1), and 2nd pleura (2) with shallow septum on abvalvar edge at apex. Scale bars: (<b>a</b>,<b>c</b>) = 10 µm, (<b>b</b>,<b>d</b>–<b>f</b>) = 5 µm.</p> Full article ">Figure 23
<p><span class="html-italic">Licmophora</span>, cont. (<b>a</b>) <span class="html-italic">L. peragallioides</span>, LM, showing apical window in septum (arrow). (<b>b</b>) <span class="html-italic">L. remulus</span>, LM, showing regular areolae in the lamina. (<b>c</b>–<b>e</b>) <span class="html-italic">L. romuli</span>, SEM, valve with details of basal pole and lamina; (<b>d</b>) showing single line of areolae on each side of the sternum on the “stem” with short striae on the basal pole, and 8 slits in the multiscissura; (<b>e</b>) showing the centripetal loss of vimines on the lamina, resulting in shredding of the valve in acid cleaning. (<b>f</b>) <span class="html-italic">L. undulata</span>, LM, showing undulation (arrows), parallel sides of lower stem, and inflated base. Scale bars: (<b>c</b>) = 25 µm, (<b>a</b>,<b>b</b>,<b>e</b>,<b>f</b>) = 10 µm, (<b>d</b>) = 2 µm.</p> Full article ">Figure 24
<p>(<b>a</b>,<b>b</b>) <span class="html-italic">Podocystis adriatica</span>. (<b>a</b>) SEM, external, showing bi- to multiseriate striae. (<b>b</b>) LM showing costae between the striae. (<b>c</b>) <span class="html-italic">Podocystis spathulata</span> SEM, internal, showing absence of costae; this valve has apical and basal rimoportulae (arrowheads), the other valve would have only apical. There is also a characteristic pore near the sternum (arrow). (<b>d</b>,<b>e</b>) <span class="html-italic">Lioloma delicatulum</span>, SEM external valve faces, showing basal pole with rimoportula (arrowhead) and part of the middle of a very long valve. (<b>f</b>–<b>h</b>) <span class="html-italic">Lioloma elongatum</span>, SEM external valve faces, showing basal pole with rimoportula (arrowhead) and portion of the middle with a “bubble-shaped structure” (arrow). Scale bars: (<b>a</b>–<b>c</b>) = 10 µm, (<b>d</b>–<b>f</b>,<b>h</b>) = 5 µm, (<b>g</b>) = 2 µm.</p> Full article ">Figure 25
<p>(<b>a</b>) <span class="html-italic">Thalassionema synedriforme</span>, SEM portion with apical spine. (<b>b</b>,<b>c</b>) <span class="html-italic">Thalassionema baculum</span>. (<b>b</b>). Frustule in LM. (<b>c</b>) Frustules in girdle view showing simple bars across areolae. (<b>d</b>–<b>g</b>) <span class="html-italic">Thalassiothrix gibberula</span>. (<b>d</b>) Portion of valve in LM showing prominent spines. (<b>e</b>) Basal pole with characteristic spines, SEM. (<b>f</b>) Portions of valves showing interior and exterior. (<b>g</b>) Fragments of valve in external view. Scale bars: (<b>b</b>,<b>d</b>,<b>f</b>) = 10 µm, (<b>a</b>,<b>c</b>,<b>e</b>,<b>g</b>) = 5 µm.</p> Full article ">Figure 26
<p>(<b>a</b>,<b>b</b>) <span class="html-italic">Gato hyalinus</span>, LM showing hyaline valve face with apical rimoportula (arrow), and SEM external valve face showing very fine striae and opening of apical rimoportula (arrow). (<b>c</b>–<b>g</b>) <span class="html-italic">Glyphodesmis acus</span>. (<b>c</b>–<b>e</b>) Yap specimens in LM (same scale). (<b>f</b>) Guam specimen (GU52P-7) in LM. (<b>g</b>) Yap specimen in SEM, internal face and narrow copulae. Scale bars: (<b>a</b>–<b>f</b>) = 10 µm, (<b>g</b>) = 5 µm.</p> Full article ">Figure 27
<p><span class="html-italic">Glyphodesmis acus</span>, cont., Guam specimens (GU52P-7), SEM. (<b>a</b>) Valve, internal view. (<b>b</b>) Frustule, oblique internal view, showing interlocking spines between two valves, multiple copulae (some hyaline, others perforate), and elevated center. (<b>c</b>) Detail of same specimen to show apical pore fields (arrows). (<b>d</b>) Portion of different frustule in girdle view near center, showing copulae and overlapping spines. Scale bars: (<b>a</b>,<b>b</b>) = 10 µm, (<b>d</b>) = 5 µm, (<b>c</b>) = 2 µm.</p> Full article ">Figure 28
<p>(<b>a</b>,<b>b</b>) <span class="html-italic">Colliculoamphora gabgabensis</span>, frustule in SEM and valve in LM. (<b>c</b>) <span class="html-italic">Lyrella clavata</span>, LM. (<b>d</b>,<b>e</b>) <span class="html-italic">Lyrella</span> cf. <span class="html-italic">rudiformis</span>, SEM, valve exterior and detail of areolae. (<b>f</b>) <span class="html-italic">Lyrella lyra</span>, L.M. (<b>g</b>) <span class="html-italic">Lyrella clavata</span> valve exterior, SEM. Scale bars: (<b>a</b>,<b>c</b>,<b>f</b>,<b>g</b>) = 10 µm, (<b>d</b>) = 5 µm, (<b>b</b>) = 2 µm, (<b>e</b>) = 1 µm.</p> Full article ">Figure 29
<p>(<b>a</b>–<b>c</b>) <span class="html-italic">Moreneis</span> cf. <span class="html-italic">hexagona</span>. (<b>a</b>) Valve in LM. (<b>b</b>) Valve in SEM, external view showing characteristic central raphe endings (arrow). (<b>c</b>) Broken frustule in SEM showing interior covered foramina. Also note the single bar extending into each areola (arrow). (<b>d</b>) <span class="html-italic">Petroneis granulata</span>, LM. (<b>e</b>–<b>g</b>) <span class="html-italic">Petroneis humerosa</span>. (<b>e</b>) Valve in LM. (<b>f</b>) Frustule in SEM, external also showing copulae. (<b>g</b>) Valve internal surface in SEM. Scale bars: (<b>a</b>,<b>d</b>–<b>g</b>) = 10 µm, (<b>b</b>,<b>c</b>) = 5 µm.</p> Full article ">Figure 30
<p>(<b>a</b>) <span class="html-italic">Mastogloiopsis biseriata</span>, frustule in girdle view, SEM. (<b>b</b>,<b>c</b>) <span class="html-italic">Tetramphora decussata</span> internal view in SEM, valve in LM. (<b>d</b>) <span class="html-italic">Tetramphora intermedia</span>, LM. (<b>e</b>,<b>f</b>) <span class="html-italic">Dictyoneis</span> cf. <span class="html-italic">marginata</span>, frustules in girdle and oblique views, LM. Scale bars: (<b>b</b>–<b>d</b>) = 25 µm, (<b>e</b>,<b>f</b>) = 10 µm, (<b>a</b>) = 5 µm.</p> Full article ">Figure 31
<p>(<b>a</b>) <span class="html-italic">Gomphonemopsis littoralis</span>, SEM. (<b>b</b>,<b>c</b>) <span class="html-italic">Achnanthes armillaris</span>. (<b>b</b>) Internal view of raphe valve showing costae and stauros forked under mantle (arrow). (<b>c</b>) Girdle view of raphe valve and cingulum showing mantle areolae in stauros fork (arrow). (<b>d</b>,<b>e</b>) <span class="html-italic">Achnanthes</span> cf. <span class="html-italic">brevipes</span>. (<b>d</b>) Internal view of raphe valve showing flat stauros and lack of costae. (<b>e</b>) Frustule in girdle view showing mantle rim with apical spines of sternum valve. (<b>f</b>–<b>h</b>) <span class="html-italic">Achnanthes kuwaitensis</span>. (<b>f</b>) Internal view of sternum valve. (<b>g</b>) External detail of sternum valve showing apical obiculus (cribrum mostly missing). (<b>h</b>) Internal detail of sternum valve apex with intact cribrum in obiculus. (<b>i</b>) <span class="html-italic">Achnanthes parvula</span>, Yap voucher, SEM (courtesy of Nelson Navarro). Scale bars: (<b>c</b>–<b>f</b>) = 10 µm, (<b>b</b>,<b>g</b>) = 5 µm, (<b>a</b>,<b>h</b>,<b>i</b>) = 2 µm.</p> Full article ">Figure 32
<p><span class="html-italic">Achnanthes</span> cf. <span class="html-italic">brevipes</span>, Pohnpei population (PN1-1). (<b>a</b>) General view of population on filamentous seaweed, frustules attached by raphe valves. (<b>b</b>) Frustule in girdle view showing rim with spines on sternum valve. Scale bars: (<b>a</b>) = 50 µm, (<b>b</b>) = 10 µm.</p> Full article ">Figure 33
<p>(<b>a</b>) <span class="html-italic">Achnanthes</span> (undescribed sp.?) frustule from Yap in girdle view, showing spines and large areolae on mantle (cribrum lost on left, obscured on right). (<b>b</b>) <span class="html-italic">A. inflata</span> (freshwater) showing difference in copulae as well as the inflated center; SV = sternum valve, RV = raphe valve (Yap voucher, courtesy of N. Navarro). Scale bars: (<b>b</b>) = 10 µm, (<b>a</b>) = 5 µm.</p> Full article ">Figure 34
<p><span class="html-italic">Achnanthes grunowii</span>. (<b>a</b>) Raphe valve in LM. (<b>b</b>,<b>c</b>) Valves in SEM (Palau specimens, PW2009-22). (<b>b</b>) Sternum valve interior with valvocopula. (<b>c</b>) Raphe valve exterior (with <span class="html-italic">Diploneis crispa</span>). Scale bars: 10 µm.</p> Full article ">Figure 35
<p><span class="html-italic">Achnanthes orientalis</span>. (<b>a</b>) Exterior views of rostrate raphe and sternum valves (Palau, PW46). (<b>b</b>–<b>d</b>) Yap specimens with more typical apices in LM and SEM. (<b>e</b>) Part of frustule in oblique view showing apical curvature of sternum valve (Chuuk, TK1A). Scale bars: (<b>a</b>–<b>d</b>) = 10 µm, (<b>e</b>) = 5 µm.</p> Full article ">Figure 36
<p>(<b>a</b>–<b>c</b>) <span class="html-italic">Planothidium delicatulum</span>, SEM: raphe valve (RV) interior and exterior, sternum valve interior. (<b>d</b>–<b>f</b>) <span class="html-italic">Anorthoneis</span> sp. (<b>d</b>) Sternum valve (SV) in LM. (<b>e</b>,<b>f</b>) SV in SEM, with and without papillae. (<b>g</b>) <span class="html-italic">Cocconeis convexa</span>, RV and SV in LM. Scale bars: (<b>d</b>,<b>g</b>) = 10 µm, (<b>e</b>,<b>f</b>) = 5 µm, (<b>a</b>–<b>c</b>) = 2 µm.</p> Full article ">Figure 37
<p>(<b>a</b>,<b>b</b>) <span class="html-italic">Cocconeis convexa</span>, cont. (<b>a</b>) Sternum valve, exterior, SEM. (<b>b</b>) Raphe valve exterior (Majuro), showing convexity (whole frustule is curved to fit on algal filaments, so this lower surface is concave). (<b>c</b>) <span class="html-italic">Cocconeis coronatoides</span>, sternum valve, SEM. (<b>d</b>,<b>e</b>) <span class="html-italic">Cocconeis dirupta</span>, LM of RV and SV, respectively. (<b>f</b>) <span class="html-italic">Cocconeis heteroidea</span> frustule at two focal planes in LM, showing RV and SV. (<b>g</b>,<b>h</b>) <span class="html-italic">Berkeleya rutilans.</span> Scale bars: (<b>f</b>) = 10 µm, (<b>a</b>–<b>e</b>,<b>g</b>,<b>h</b>) = 5 µm.</p> Full article ">Figure 38
<p>(<b>a</b>,<b>b</b>) <span class="html-italic">Parlibellus biblos</span>, LM and internal SEM. (<b>c</b>,<b>d</b>) <span class="html-italic">Parlibellus hamulifer</span>, LM and internal SEM. (<b>e</b>) <span class="html-italic">Parlibellus waabensis</span>, SEM of Palau specimen, girdle view showing spacing of central striae and larger pores on copulae near mid-cell. Scale bars: (<b>a</b>,<b>c</b>–<b>e</b>) = 10 µm, (<b>b</b>) = 5 µm.</p> Full article ">Figure 39
<p><span class="html-italic">Parlibellus waabensis</span> from Pohnpei (PN1-1) (<b>a</b>,<b>c</b>–<b>e</b>) and Palau (PW(2021)4-7) (<b>b</b>). (<b>a</b>) Preserved cells in mucilage tube, LM. (<b>b</b>,<b>c</b>) Frustules in girdle view showing the distinctive large pores in the girdle bands (arrows), LM. (<b>d</b>) SEM of frustule in mucilage tube, the distinctive copula pores and wider striae spacing on center of cell visible through the dried mucilage (arrows). (<b>e</b>) Girdle view of Pohnpei valve in SEM, showing slight differences from the Palau specimen in <a href="#diversity-17-00034-f038" class="html-fig">Figure 38</a>e, especially in the valve areolae and central striae. Scale bars: (<b>a</b>) = 20 µm, (<b>b</b>–<b>e</b>) = 10 µm.</p> Full article ">Figure 40
<p><span class="html-italic">Progonoia</span> spp. (<b>a</b>) <span class="html-italic">Progonoia diatreta</span>, LM. (<b>b</b>,<b>c</b>) <span class="html-italic">Progonoia intercedens</span>. Scale bars = 10 µm.</p> Full article ">Figure 41
<p>(<b>a</b>) <span class="html-italic">Caloneis egena</span>, LM. (<b>b</b>) <span class="html-italic">Caloneis ophiocephala</span>, LM. (<b>c</b>,<b>d</b>) <span class="html-italic">Caloneis</span> cf. <span class="html-italic">petitiana</span>, LM at two focal planes, SEM at 15 kV showing finely porous membranes over alveoli. Scale bars: (<b>a</b>–<b>c</b>) = 10 µm, (<b>d</b>) = 5 µm.</p> Full article ">Figure 42
<p><span class="html-italic">Diploneis cerebrum</span>. (<b>a</b>) LM. (<b>b</b>) External valve surface, SEM. (<b>c</b>) Interior valve surface, again showing the longitudinal rib. (<b>d</b>) Detail of central part of valve with detail of “brain-like” cribra over longitudinal canal areolae; double-headed arrow shows position of longitudinal rib, arrowhead the small wave in the raphe near the central endings and single arrow the central areolae over the canal (Chuuk specimen, TK28). Scale bars: (<b>a</b>–<b>c</b>) 10 µm, (<b>d</b>) = 5 µm.</p> Full article ">Figure 43
<p><span class="html-italic">Diploneis</span> spp. (<b>a</b>) <span class="html-italic">D. chersonensis</span>, half of valve in SEM, showing flaps on raphe near central area (arrow). (<b>b</b>) <span class="html-italic">D. claustra</span>. (<b>c</b>,<b>d</b>) <span class="html-italic">D. crabro</span>, nominate variety. (<b>c</b>) LM at two focal planes showing small lunula area (arrow) and large internal foramina (arrowhead). (<b>d</b>) Internal SEM showing large foramina. (<b>e</b>) <span class="html-italic">D. crabro</span> var. <span class="html-italic">excavata</span>, LM, showing wide, excavated lunula area (arrow). Scale bars: (<b>c</b>–<b>e</b>) = 10 µm, (<b>a</b>,<b>b</b>) = 5 µm.</p> Full article ">Figure 44
<p><span class="html-italic">Diploneis denticulata</span> sp. nov., SEM except (<b>a</b>). Yap specimens from Y34A and (<b>f</b>) Y26C. (<b>a</b>) Holotype specimen at two focal planes in LM. (<b>b</b>) Exterior valve. (<b>c</b>) Half of same valve with natural tilt to show surface relief. (<b>d</b>) Oblique view of frustule, apparently in division, showing toothed rim of epitheca and inner face of daughter hypotheca. (<b>e</b>) Detail of inner valve surface with central raphe endings and intact hymenes covering striae. (<b>f</b>) Girdle view of intact frustule showing flat raphe keel, vertical mantle and hyaline cingulum. (<b>g</b>) Fragment of broken valve including central nodule, showing chambering in the longitudinal canal (arrow). (<b>h</b>) Half of valve of rounded form. Scale bars: (<b>a</b>) = 10 µm, (<b>b</b>–<b>d</b>,<b>f</b>,<b>h</b>) = 5 µm, (<b>e</b>,<b>g</b>) = 2 µm.</p> Full article ">Figure 45
<p><span class="html-italic">Diploneis</span> spp. (<b>a</b>) <span class="html-italic">D. craticula</span>, SEM exterior. (<b>b</b>–<b>d</b>) <span class="html-italic">D. papula</span>, external views of frustules, oblique view showing domed cribra of areolae between virgae (arrow). (<b>e</b>) <span class="html-italic">D. nitescens</span>, SEM of valve exterior. (<b>f</b>) <span class="html-italic">D. smithii</span>, SEM of valve exterior. (<b>g</b>) <span class="html-italic">D. smithii</span> var. <span class="html-italic">rhombica</span>, LM. (<b>h</b>) <span class="html-italic">D. suborbicularis</span>, LM. Scale bars: (<b>e–h</b>) = 10 µm, (<b>a–d</b>) = 5 µm.</p> Full article ">Figure 46
<p>(<b>a</b>–<b>d)</b> <span class="html-italic">Diploneis weissflogii</span> vs. <span class="html-italic">D. weissflogiopsis</span>, SEM. (<b>a</b>,<b>b</b>) <span class="html-italic">D. weissflogii.</span> (<b>a</b>) Internal view showing single foramen on each side of the central area (in rectangle), foramina at apex of canals (arrows), and hyaline copulae. (<b>b</b>) External valve face showing single modified stria between central raphe endings (in rectangle). (<b>c</b>,<b>d</b>) <span class="html-italic">D. weissflogiopsis</span>. (<b>c</b>) Valve showing three modified striae between raphe endings (in rectangle); the higher striae density is also evident in the comparison. (<b>d</b>) Detail of central area in slightly oblique view with the central dimple more evident (arrow). (<b>e</b>) <span class="html-italic">Navicula consors</span>. SEM. Scale bars: (<b>e</b>) =10 µm, (<b>a</b>–<b>d</b>) = 5 µm.</p> Full article ">Figure 47
<p><span class="html-italic">Cymatoneis</span> spp., SEM. (<b>a</b>,<b>b</b>) <span class="html-italic">C. sulcata</span>, Palau specimens (PW46). Valve and girdle views showing strongly developed longitudinal ribs and hyaline cingulum. (<b>c</b>) <span class="html-italic">C. belauensis</span>, sp. nov. from Palau (PW46) valve view, showing longitudinal ridges with hyaline outer sides and ribs enclosing much of raphe. (<b>d</b>). Same valve naturally tilted, showing elevations of the valve. Scale bars: (<b>a</b>–<b>c</b>) = 10 µm, (<b>d</b>) = 5 µm.</p> Full article ">Figure 48
<p><span class="html-italic">Cymatoneis</span> spp., cont. (<b>a</b>–<b>c</b>) <span class="html-italic">C. belauensis</span>, internal aspects of a valve. (<b>a</b>) Valve as found, showing valve depth from outside and outer zone of the striae. (<b>b</b>,<b>c</b>) Same valve tilted 45°; (<b>b</b>) showing whole valve, (<b>c</b>) detail of central area with small central nodule. (<b>d</b>) <span class="html-italic">C. yapensis</span>, sp. nov. from Yap (Y34A). Scale bars: (<b>a</b>,<b>b</b>,<b>d</b>) = 10 µm, (<b>c</b>) = 5 µm.</p> Full article ">Figure 49
<p>(<b>a</b>,<b>b</b>) <span class="html-italic">Navicula plicatula</span> SEM external and internal aspects of valve. (<b>c</b>) <span class="html-italic">Navicula tsukamotoi</span> valve external, SEM. Scale bars = 10 µm.</p> Full article ">Figure 50
<p>(<b>a</b>) <span class="html-italic">Trachyneis aspera</span>. (<b>b</b>) <span class="html-italic">Trachyneis velata</span>. (<b>c</b>–<b>g</b>) <span class="html-italic">Plagiotropis lepidoptera</span>. (<b>c</b>) LM at two focal planes. (<b>d</b>,<b>e</b>) SEM of external valve faces showing major and minor sides and paired areolae (arrow, <a href="#diversity-17-00034-f050" class="html-fig">Figure 50</a>e). (<b>f</b>,<b>g</b>) Central area with enlargement of central nodule. Scale bars: (<b>a</b>–<b>d</b>) = 10 µm, (<b>f</b>) = 5 µm, (<b>e</b>) = 2 µm, (<b>g</b>) = 1 µm.</p> Full article ">Figure 51
<p>Morphological characters in <span class="html-italic">Plagiotropis</span> across Micronesia. (<b>a</b>) Unidentified species with scuta (arrows) (Y34H). (<b>b</b>) Portion of a valve showing double wall with external silica flap (arrow) forming the scutum (PW1990-47). (<b>c</b>) Portion of a valve showing single wall pleated to produce the scutum (arrow) (GU68D-1B). (<b>d</b>) Specimen with conopeum over central raphe endings (arrow) (Y45-2). Scale bars: (<b>a</b>,<b>d</b>) = 10 µm, (<b>b</b>,<b>c</b>) = 2 µm.</p> Full article ">Figure 52
<p>(<b>a</b>,<b>b</b>) <span class="html-italic">Pleurosigma simulacrum</span>, Yap voucher specimen in SEM at two magnifications. (<b>c</b>,<b>d</b>) <span class="html-italic">Rhoicosigma parvum</span>. Convex valve with straight raphe in LM, concave valve with sigmoid raphe in SEM. (<b>e</b>–<b>g</b>) <span class="html-italic">Schizostauron</span> cf. <span class="html-italic">trachyderma</span>. (<b>e</b>) Raphe valve in LM. (<b>f</b>) Sternum valve in LM. (<b>g</b>) Sternum valve internal aspect in SEM. Scale bars: (<b>a</b>) = 20 µm, (<b>c</b>–<b>g</b>) = 10 µm, (<b>b</b>) = 5 µm.</p> Full article ">Figure 53
<p><span class="html-italic">Amphora</span> spp. (<b>a</b>) <span class="html-italic">A. arenaria</span>, LM. (<b>b</b>,<b>c</b>) <span class="html-italic">A. bigibba</span> SEM of frustules, arrows showing rows of apically elongate areolae near raphe ledge. (<b>d</b>,<b>e</b>) <span class="html-italic">A. bigibba</span> var. <span class="html-italic">interrupta</span>. SEM of frustule in ventral view and isolated valve, external aspect. Scale bars: (<b>a</b>) = 10 µm, (<b>b</b>–<b>e</b>) = 5 µm.</p> Full article ">Figure 54
<p>(<b>a</b>) <span class="html-italic">Amphora obtusa</span>, LM. (<b>b</b>) <span class="html-italic">Amphora immarginata</span>, SEM valve external. (<b>c</b>) <span class="html-italic">Amphora ostrearia</span> var. <span class="html-italic">vitrea</span>, SEM valve external. (<b>d</b>,<b>e</b>) <span class="html-italic">Amphora proteus</span>, LM, vale and frustule. (<b>f</b>) <span class="html-italic">Amphora spectabilis</span>, LM. (<b>g</b>) <span class="html-italic">Amphora subhyalina</span>, SEM of frustule. Scale bars = 10 µm.</p> Full article ">Figure 55
<p>(<b>a</b>,<b>b</b>) <span class="html-italic">Thalassiophysa hyalina</span>. (<b>a</b>) Valve in LM. (<b>b</b>) Center of valve exterior in SEM. (<b>c</b>) <span class="html-italic">Undatella lineata</span> valve in LM. (<b>d</b>) “<span class="html-italic">Bacillaria paradoxa</span>” Group B, SEM showing characteristic T-shaped terminal raphe ending (arrow). (<b>e</b>,<b>f</b>) <span class="html-italic">Cymatonitzschia marina</span>, frustules in LM and SEM. Scale bars: (<b>a</b>,<b>c</b>,<b>e</b>,<b>f</b>) = 10 µm, (<b>b</b>,<b>d</b>) = 5 µm.</p> Full article ">Figure 56
<p><span class="html-italic">Homoeocladia</span> spp. Yap vouchers, SEM. (<b>a</b>) <span class="html-italic">H. schefteropsis</span>, showing areolae in peri-raphe zone (black arrows) and squiggly areolae on copulae (white arrow). (<b>b</b>) <span class="html-italic">H. coacervata</span>, showing areolae in peri-raphe zone (black arrow) and stacked slits on copulae (white arrow). (<b>c</b>) <span class="html-italic">H. micronesica</span>, showing no areolae in peri-raphe zone (black arrow). A row of small pores is present on the apex in all three species (arrowheads). Scale bars = 2 µm.</p> Full article ">Figure 57
<p><span class="html-italic">Homoeocladia</span> spp., SEM. (<b>a</b>,<b>b</b>) <span class="html-italic">H. martiana</span> valve fragment at two magnifications. (<b>c</b>,<b>d</b>) <span class="html-italic">H. volvendirostrata</span>, frustule at two magnifications. Scale bars: (<b>a</b>) = 25 µm, (<b>b</b>,<b>c</b>) = 5 µm, (<b>c</b>) = 2 µm.</p> Full article ">Figure 58
<p><span class="html-italic">Nitzschia</span> spp. (<b>a</b>–<b>c</b>) <span class="html-italic">Nitzschia longissima</span>, SEM. (<b>a</b>,<b>b</b>) Whole valve, external view, with detail of central portion. (<b>c</b>) Internal aspect near central area showing uninterrupted striae and character of fibulae. (<b>d</b>,<b>e</b>) <span class="html-italic">N. maiae</span>, SEM and LM of frustule. (<b>f</b>) <span class="html-italic">N. marginulata</span> var. <span class="html-italic">didyma</span> frustule SEM. Scale bars: (<b>a</b>) = 25 µm, (<b>b</b>–<b>f</b>) = 10 µm.</p> Full article ">Figure 59
<p><span class="html-italic">Nitzschia pseudohybridopsis</span>, sp. nov. (<b>a</b>–<b>c</b>) Range of size in LM, (<b>c</b>) = holotype. (<b>d</b>) Frustule in girdle view, SEM, showing ‘papillae’ on valvocopula. (<b>e</b>) Valve in valve view. Scale bars: (<b>a</b>–<b>c</b>) = 10 µm, (<b>d</b>,<b>e</b>) = 5 µm.</p> Full article ">Figure 60
<p><span class="html-italic">Nitzschia pseudohybridopsis</span>, sp. nov., cont. (<b>a</b>,<b>b</b>) SEM of frustules showing details of the valvocopula (VC) with small pars exterior (pe) and larger papillate pars interior (pi), (<b>b</b>) also showing part of the internal keel with central nodule. Scale bars: 5 µm.</p> Full article ">Figure 61
<p>(<b>a</b>) <span class="html-italic">Nitzschia ventricosa</span>, valve fragment, LM. (<b>b</b>) <span class="html-italic">Psammodictyon constrictum</span> frustule in SEM. (<b>c</b>) <span class="html-italic">Psammodictyon pustulatum</span> frustule in SEM. (<b>d</b>,<b>e</b>) <span class="html-italic">Psammodictyon panduriforme</span>, SEM. (<b>d</b>) Frustule showing exterior valve face. (<b>e</b>) Internal valve face. Scale bars: (<b>a</b>,<b>d</b>,<b>e</b>) = 10 µm, (<b>b</b>,<b>c</b>) = 5 µm.</p> Full article ">Figure 62
<p>(<b>a</b>) <span class="html-italic">Tryblionella granulata</span>, frustule, SEM, Yap voucher, courtesy of Nelson Navarro. (<b>b</b>–<b>g</b>) <span class="html-italic">Entomoneis yudinii</span>, sp. nov. (<b>b</b>,<b>c</b>) Valves in valve view, LM; (<b>c</b>) = holotype. (<b>d</b>) Valve in valve view showing overall shape and features of exterior. (<b>e</b>) Detail of valve showing depressed area with infilled areolae and the faint asymmetrical stauros (arrows). (<b>f</b>) Detail of valve in (<b>d</b>) showing outer coverings of areolae and a grooved rib alongside the raphe slit (arrow). (<b>g</b>) Interior showing smaller inner foramina, also occluded, and part of the keel canal with fibulae (arrow). Scale bars: (<b>b</b>–<b>d</b>) = 10 µm, (<b>a</b>,<b>e</b>,<b>g</b>) = 5 µm, (<b>f</b>) = 2 µm.</p> Full article ">Figure 63
<p><span class="html-italic">Epithemia</span> spp. in SEM. (<b>a</b>–<b>c</b>) <span class="html-italic">E. guettingeri</span>. (<b>a</b>) Valve view of ventral surface, showing axial “costa” formed by narrow wave in surface (arrow), note areolae in the groove (arrowhead). Stria density about 30 along keel. (<b>b</b>) Small specimen, ca. 40 striae, axial costa not so clear. (<b>c</b>) Interior aspect of valve showing transverse costae and part of keel canal (Palau specimen PW(2022)1A-5). (<b>d</b>,<b>e</b>) <span class="html-italic">Epithemia muscula</span>, SEM. (<b>d</b>) Frustule in oblique dorsal view, showing narrow dorsal surface (arrow). (<b>e</b>) Interior aspect of valve showing numerous transverse costae and large cribrate areolae. Scale bars = 5 µm.</p> Full article ">Figure 64
<p>(<b>a</b>,<b>b</b>) <span class="html-italic">Protokeelia cholnokyi</span> valve in LM and frustule in SEM. (<b>c</b>) <span class="html-italic">Auricula intermedia</span>. (<b>d</b>) <span class="html-italic">Campylodiscus giffenii</span> valve, SEM. (<b>e</b>) <span class="html-italic">Campylodiscus humilis</span> valve, LM. Scale bars: (<b>a</b>,<b>c</b>,<b>e</b>) = 10 µm, (<b>b</b>,<b>d</b>) = 5 µm.</p> Full article ">Figure 65
<p><span class="html-italic">Campylodiscus tatreauae</span> sp. nov. Yap (<b>a</b>–<b>c</b>) and Palau (<b>d</b>–<b>g</b>). (<b>a</b>) Holotype in LM showing barred fenestrae (arrow). (<b>b</b>) Valve exterior showing aqueduct-like rim (double-headed arrow), silica flaps, and barred fenestrae (arrow). (<b>c</b>,<b>d</b>) Details of margin with barred fenestrae (arrows) (<b>c</b>) in LM from another specimen on same slide, (<b>d</b>) in SEM (PW(2022)1A-5). (<b>e</b>) Valve interior. (<b>f</b>) Detail of valve interior (same specimen). (<b>g</b>) Oblique external view of valve showing rim (double-headed arrow) and silica flaps. Scale bars: (<b>a</b>–<b>c</b>,<b>e</b>) = 10 µm, (<b>d</b>,<b>f</b>,<b>g</b>) = 5 µm.</p> Full article ">Figure 66
<p>(<b>a</b>) <span class="html-italic">Campylodiscus neofastuosus</span>, valve in SEM, showing small fenestrae and “pie-crust” rim (arrows). (<b>b</b>) <span class="html-italic">Coronia ambigua</span>, LM. (<b>c</b>) <span class="html-italic">Coronia decora</span> var. <span class="html-italic">decora</span>, LM, frustule showing two axes at right angles. (<b>d</b>) <span class="html-italic">Coronia decora</span> var. <span class="html-italic">pinnata</span> valve, LM. (<b>e</b>,<b>f</b>) <span class="html-italic">Hydrosilicon mitra</span>, SEM. (<b>e</b>) Yap voucher. (<b>f</b>) Detail of middle of valve interior, Guam specimen, showing striae converging on transverse and longitudinal ribs (see also <a href="#diversity-17-00034-f067" class="html-fig">Figure 67</a>a,b). Scale bars: (<b>a</b>–<b>e</b>) = 10 µm, (<b>f</b>) = 5 µm.</p> Full article ">Figure 67
<p>(<b>a</b>,<b>b</b>) <span class="html-italic">Hydrosilicon mitra</span>, Guam specimen, showing entire specimen, valve internal view, and higher magnification of constriction where the two raphe endings meet in the nodule and from whence the striae radiate towards the transverse and longitudinal costae. Scale bars: (<b>a</b>) = 10 µm, (<b>b</b>) = 5 µm.</p> Full article ">Figure 68
<p><span class="html-italic">Petrodictyon.</span> (<b>a</b>–<b>c</b>) <span class="html-italic">Petrodictyon gemma</span>. (<b>a</b>) LM showing visible striae and areolae. (<b>b</b>) Half of external valve view, SEM. (<b>c</b>) Half of internal view, SEM, inset showing 2–3 portulae between fibulae. (<b>d</b>–<b>f</b>) <span class="html-italic">Petrodictyon patrimonii</span>. Fig. (<b>d</b>) LM showing striae not resolved. (<b>e</b>) Internal view, SEM, inset showing 3–6 portulae between fibulae. (<b>f</b>) Frustule (broken open), showing depth of mantle. Scale bars = 10 µm.</p> Full article ">

Figure 1
<p>Model of TLK1B mTOR-driven translational derepression, followed by phosphorylation and activation of some of its key targets.</p> Full article ">

Figure 1
<p>Modulation of CTTN expression during myoblast differentiation. (<b>A</b>) Immunoblotting was conducted to assess CTTN expression levels in C2C12 myoblasts and various tissues from C57BL/6 mice, with α-tubulin as a loading control. (<b>B</b>) C2C12 myoblasts were harvested on specified differentiation days, and the protein expression levels of MyoD, MyoG, MyHC, and CTTN were analyzed by immunoblotting, with β-actin as a loading control. (<b>C</b>) Protein expression levels were normalized to β-actin, and relative expression ratios were calculated, setting day 0 as one for MyoD and CTTN, day 1 for MyoG, and day 2 for MyHC. Data are presented as means ± SEM (n = 3), with asterisks indicating statistical significance (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 2
<p>CTTN knockdown led to a reduction in F-actin levels and an increase in G-actin levels. C2C12 myoblasts were transfected with 200 nM of either control scRNA or siCTTN (siCTTN-1 or siCTTN-2). (<b>A</b>) CTTN expression was assessed by immunoblotting 24 h after transfection. CTTN expression levels were normalized to β-actin, and relative expression ratios were calculated with the control scRNA set to one. (<b>B</b>) After 24 h post-transfection, cells were stained with FITC-phalloidin (green) for F-actin and Hoechst 33,342 (blue) for nuclei. Scale bar: 25 μm. Phalloidin intensities were quantified using ImageJ software, version 1.5.4. (<b>C</b>) F- and G-actin levels were quantified by flow cytometry after staining with FITC-phalloidin for F-actin and DNase I for G-actin, respectively. Data are presented as means ± SEM (n = 3), with asterisks indicating statistical significance (** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 3
<p>CTTN depletion impaired the nuclear localization of MRTFA and YAP1. C2C12 myoblasts were transfected with either control scRNA or siCTTN and analyzed 24 h post-transfection. (<b>A</b>) Cytoplasmic and nuclear fractions were subjected to immunoblot analysis for MRTFA, SRF, YAP1, pYAP1 (phosphorylated YAP1), and CTTN expression. For MRTFA, different exposure times were used to account for its varied distribution between cytoplasmic and nuclear compartments. α-Tubulin and lamin B2 served as cytoplasmic and nuclear markers, respectively. β-Actin was used as a loading control. (<b>B</b>,<b>C</b>) The protein expression levels were normalized to β-actin, and relative expression ratios were calculated with the control scRNA set to one. Data are presented as means ± SEM (n = 3), with asterisks indicating statistical significance (** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 4
<p>CTTN knockdown suppressed SRF transcriptional activity. (<b>A</b>) Diagram of the luciferase reporter construct featuring the truncated SMYD1 promoter region, including the CArG box for SRF binding. (<b>B</b>) C2C12 myoblasts were transfected with either the pGL3 vector (Vector) or pGL3 containing the SMYD1 promoter (SMYD1) along with control scRNA or siCTTN. Relative luciferase activity was measured 24 h post-transfection. (<b>C</b>) C2C12 myoblasts were transfected with either control scRNA or siCTTN, and mRNA levels of SRF, Vinculin, and SMYD1 were assessed by RT-qPCR, normalized to GAPDH expression 24 h post-transfection. Data are presented as means ± SEM (n = 3), with asterisks indicating statistical significance (** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001); ns indicates non-significance.</p> Full article ">Figure 5
<p>CTTN depletion impeded cell proliferation and cell cycle progression. C2C12 myoblasts were transfected with either control scRNA or siCTTN and analyzed 24 h post-transfection. (<b>A</b>) Cell proliferation was evaluated by EdU incorporation (green) to label replicating cells, with Hoechst 33,342 (blue) as a nuclear counterstain. Scale bar: 50 µm. (<b>B</b>) The percentage of EdU-positive cells was quantified using ImageJ software. (<b>C</b>) Viable cell numbers were measured using a cell viability assay kit. (<b>D</b>) mRNA levels of proliferation markers (PCNA, cyclin B1, and cyclin D1) were assessed by RT-<span class="html-italic">q</span>PCR and normalized to GAPDH expression. (<b>E</b>,<b>F</b>) Cell cycle analysis was performed using flow cytometry with scatter plots. Data are presented as means ± SEM (n = 3), with asterisks indicating statistical significance (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 6
<p>CTTN knockdown suppressed the expression of myogenic regulatory factors. (<b>A</b>) C2C12 myoblasts were transfected with either control scRNA or siCTTN, allowed to differentiate, and then harvested on specified differentiation days. Protein expression levels of MyoD, MyoG, MyHC, and CTTN were analyzed by immunoblotting. (<b>B</b>) Protein expression levels for scRNA (open column) and siCTTN (blue column) were normalized to β-actin and presented as relative ratios, with scRNA expression levels on day 0 (for CTTN and MyoD) or day 3 (for MyoG and MyHC) set to one. Data are presented as means ± SEM (n = 3), with asterisks indicating statistical significance (* <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001); ns indicates non-significance.</p> Full article ">Figure 7
<p>CTTN depletion impaired myogenic differentiation. C2C12 myoblasts were transfected with either control scRNA or siCTTN and then allowed to differentiate for 5 days. (<b>A</b>) Representative immunocytochemistry stained with MyHC antibody (green) and Hoechst 33,342 (blue). Scale bar: 100 μm. (<b>B</b>) MyHC-positive areas, differentiation indices, fusion indices, and myotube widths were determined as described in <a href="#sec4-ijms-25-13564" class="html-sec">Section 4</a>. Data are presented as means ± SEM (n = 3), with asterisks indicating statistical significance (*** <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 8
<p>Schematic illustration of the actin-MRTFA-SRF and YAP1 signaling pathway regulated by CTTN.</p> Full article ">

Figure 1
<p>(<b>a</b>) A table summarising the data on the increase or decrease in various markers under YAP overactivation and suppression. (<b>b</b>) Top panel: in normal skin, YAP is localised in nuclei in the basal layer of the epidermis and is detected in the cytoplasm in differentiated layers. In mature HFs, YAP is mainly cytoplasmic in differentiated cells of the IRS and hair shaft and is retained in the nucleus in the hair matrix and ORS. Middle panel: YAP overactivation results in abnormal HF structure, widening of the basal layer of the epidermis, and disruption of terminal differentiation, as well as a fibrotic state of the dermis with fibroblast hyperproliferation and increased collagen production. Bottom panel: YAP suppression disrupts the HF cycle and forms a thin epidermis with abnormal stratification. Abbreviations: BL, basal layer; IRS, inner root sheath; SL, spinous layer; ORS, outer root sheath [<a href="#B14-ijms-25-12903" class="html-bibr">14</a>,<a href="#B18-ijms-25-12903" class="html-bibr">18</a>,<a href="#B19-ijms-25-12903" class="html-bibr">19</a>,<a href="#B21-ijms-25-12903" class="html-bibr">21</a>,<a href="#B22-ijms-25-12903" class="html-bibr">22</a>,<a href="#B40-ijms-25-12903" class="html-bibr">40</a>,<a href="#B41-ijms-25-12903" class="html-bibr">41</a>,<a href="#B42-ijms-25-12903" class="html-bibr">42</a>,<a href="#B49-ijms-25-12903" class="html-bibr">49</a>,<a href="#B52-ijms-25-12903" class="html-bibr">52</a>,<a href="#B53-ijms-25-12903" class="html-bibr">53</a>,<a href="#B57-ijms-25-12903" class="html-bibr">57</a>,<a href="#B59-ijms-25-12903" class="html-bibr">59</a>,<a href="#B61-ijms-25-12903" class="html-bibr">61</a>,<a href="#B63-ijms-25-12903" class="html-bibr">63</a>,<a href="#B65-ijms-25-12903" class="html-bibr">65</a>,<a href="#B66-ijms-25-12903" class="html-bibr">66</a>,<a href="#B68-ijms-25-12903" class="html-bibr">68</a>,<a href="#B71-ijms-25-12903" class="html-bibr">71</a>,<a href="#B74-ijms-25-12903" class="html-bibr">74</a>,<a href="#B75-ijms-25-12903" class="html-bibr">75</a>,<a href="#B76-ijms-25-12903" class="html-bibr">76</a>,<a href="#B77-ijms-25-12903" class="html-bibr">77</a>,<a href="#B78-ijms-25-12903" class="html-bibr">78</a>].</p> Full article ">Figure 2
<p>Mechanisms of YAP activation in KCs. The canonical Hippo signalling pathway via the kinases MST1/2 and LATS1/2 inactivate YAP/TAZ via serine phosphorylation to enhance retention in the cytoplasm and/or proteasomal degradation. Phosphorylated YAP is sequestered in the cytoplasm by a membrane protein complex that includes AJs, desmosome proteins, integrins, and their cytoplasmic adaptors. In the complex, α-catenin controls YAP/TAZ activity and phosphorylation by modulating their interaction with 14-3-3 proteins and PP2AC. DSG3 and its adaptor protein plakophilin 1 (PKP1) are also involved in the membrane complex sequestration of YAP. Plakophilin 4 (PKP4) may function as a recruiter of YAP to the junctional zone or as a mediator of the disruptor of the YAP/TAZ/SAV1/LATS1 complex. ANXA2 was identified as a Hippo pathway modulator that controls YAP phosphorylation. Keratin filaments (KRT4/15) also control YAP activation through 14-3-3σ binding. Stabilisation of F-actin with functioning myosin II and ARP2/3 is required for stiffness-induced nuclear localisation of YAP/TAZ. Integrin/Src signalling contributes to the nuclear localisation of YAP, as Src can directly phosphorylate YAP at the three binding sites, activating it. Src activation mediated by integrin α6β4 negatively regulates α-catenin, whereas integrinα3β1/Src signalling is inhibited by their interaction with THY1. Additionally, YAP/TAZ activity is regulated at the nuclear level. Under mild mechanical stress in the nucleus, YAP/TAZ are sequestered in the pool of SWI/SNF chromatin remodelling complexes containing ARID1A, which prevents YAP/TAZ from binding to TEADs.</p> Full article ">Figure 3
<p>YAP and TAZ interactions with signalling pathways and TFs. EGFR, FGFR2, IL17R, and P2RY6 drive YAP and TAZ activation. WNT pathway activation disrupts the cytoplasmic YAP sequestration complex, resulting in the release of YAP and β-catenin and their nuclear translocation. TGFBR activation transduces the signal to the SMAD2/3 complex, which interacts with YAP/TAZ and binds SMAD4 for nuclear shuttling. SMAD7 is a component of the cytoplasmic protein complex that suppresses YAP activity. In the nucleus, YAP and TAZ are recruited to distant regulatory elements, enhancers and super-enhancers, in association with TEADs and AP-1. YAP and TAZ recruit BRD4 and the Mediator complex to promote transcription. YAP/TAZ-mediated engagement of the MMB complex initiates chromatin looping and transcription of cell cycle-related genes (*). Besides the MMB complex, YAP and TAZ could be involved in chromatin remodelling via association with the SWI/SNF complex and Hippo-mediated suppression of CTCF activity. LEF/TCF, SNAI1/2, SMAD2/3, and ΔNp63 can form transcription complexes with YAP/TAZ, while VGLL4 and KLF4 suppress YAP/TAZ-mediated transcription. TAZ can form nuclear condensates via LLPS. The FUS protein associates with TAZ in nuclear condensates, maintaining their liquid state and thus facilitating TAZ-mediated transcriptional regulation.</p> Full article ">Figure 4
<p>Functioning of YAP/TAZ in the epidermis under normal and pathological conditions. Several aspects of the functioning of the YAP/TAZ currently require further investigation: the influence of VGLL on KC differentiation; the role of different TEAD isoforms in the expression of YAP/TAZ target genes; and how the organisation of protein complexes and reorganisation of chromatin structure regulate transcriptional activity.</p> Full article ">

Figure 1
<p>Schematic representation of intramolecular and intermolecular FRET biosensors. (<b>A</b>) Intramolecular FRET. A single protein with CFP and YFP attached at two ends undergoes conformational changes, bringing CFP and YFP into proximity. Energy transfer occurs when CFP is excited, leading to emission from YFP. (<b>B</b>) Intermolecular FRET. Two interacting proteins, A and B, are tagged with CFP and YFP, respectively. When the proteins interact closely, FRET occurs as energy from the donor (CFP) is transferred to the acceptor (YFP), resulting in fluorescence from YFP. These FRET configurations allow the monitoring of protein interactions and conformational changes in live cells.</p> Full article ">Figure 2
<p>Schematic presentation of TR-FRET biosensors. The target protein is bound by two antibodies: Eu-Ab1, labeled with a europium (Eu) donor fluorophore, and D2-Ab2, labeled with the D2 acceptor fluorophore. Upon excitation at 320–340 nm, the Eu donor emits at 615 nm. When the antibodies are in close proximity due to binding the same protein or two interacting proteins, FRET occurs from the Eu donor to the acceptor, resulting in a TR-FRET emission at 665 nm.</p> Full article ">Figure 3
<p>Schematic representation of the BRET and NanoBRET biosensor. (<b>A</b>) The energy donor such as a luciferase enzyme (RLuc or NanoLuc) is fused to one protein of interest (Protein A), while a fluorescent acceptor is attached to the interacting partner (Protein B). When Protein A and B interact within a close distance (~5–10 nm), upon substrate (e.g., coelenteramide) oxidation, the luciferase enzyme (donor) releases energy in the form of photons, with specific emission wavelengths (e.g., ~480 nm for RLuc). The acceptor fluorophore absorbs the donor emission and re-emits it at a higher wavelength (e.g., ~530 nm for YFP), producing a measurable light signal (<b>B</b>) when protein–protein interactions bring the donor and acceptor close together.</p> Full article ">Figure 4
<p>Schematic representation of bioluminescent biosensors using firefly SLCA monitoring PPIs. (<b>A</b>) Representation of the SLCA components in luciferase structure. (<b>B</b>) Split luciferase assays in monitoring PPIs. In this biosensor system, the firefly luciferase enzyme is split into two non-functional fragments: the N-terminal domain (Nluc, amino acids 1–416) and the C-terminal domain (Cluc, amino acids 394–550). These fragments are fused to two proteins of interest, Protein A and Protein B. When Protein A and Protein B interact, the two luciferase fragments are brought into close proximity, allowing them to reconstitute the functional enzyme. (<b>C</b>) Luciferase assays. In the presence of the substrate D-luciferin and cofactors such as Mg²⁺, the restored luciferase catalyzes the oxidation of D-luciferin to oxyluciferin, producing light. This luminescence signal indicates the interaction between the two proteins and can be quantified to measure the strength and dynamics of the PPIs.</p> Full article ">Figure 5
<p>Schematic representation of the NanoBiT biosensors. The NanoBiT system utilizes two complementary luciferase fragments, LgBiT and SmBiT, which are fused to proteins of interest (Protein A and Protein B). Upon interaction between the two proteins, the fragments reassemble to form an active NanoLuc luciferase, generating bioluminescence in the presence of the substrate furimazine.</p> Full article ">

Figure 1
<p>Experimental design. Bone marrow cells isolated from Balb/C femur and tibia were cultured in 25 cm² flasks for 4 days according to the routine protocol. On day 5, the medium containing non-adherent cells was removed and the flasks were completely filled with a fresh culture medium—intact medium (int) and osteogenic culture medium (ost). Heterotypic bone marrow cell cultures were divided into 2 groups: 1 g—standard culture conditions; and smg—3D clinorotation for 14 days. At the end of the experiment, the cells were initiated for in vitro analysis.</p> Full article ">Figure 2
<p>Heterotypic cell cultures of murine bone marrow after 14 days of in vitro exposure to 1 g (<b>left</b> column) or smg (<b>right</b> column). Top 4 images show intact cells, and bottom 4 images are osteoinduced cells. Phase contrast microscopy, scale bar—100 μm. Abbreviations: int (intact medium)—complete culture medium; ost—osteogenic culture medium; 1 g—standard culture conditions; smg—3D clinorotation for 14 days.</p> Full article ">Figure 3
<p>Flow cytometry analysis of intact and osteoinduced heterotypic cell culture from murine bone marrow under 1 g and smg conditions. Heterotypic cell culture viability analysis: (<b>a</b>) representative density plots of the heterotypic cell culture double-stained with PI and Annexin V; (<b>b</b>) the proportion of viable cells in the population, defined as PI-Annexin V-, n = 5, ns - not significant; (<b>c</b>) the proportion of viable cells in the CD45+ and CD45− populations, defined as 7AAD-negative cells, n = 5. Determination of the ratio of hematopoietic and stromal compartments in heterocellular culture: (<b>d</b>) representative histograms of CD45 staining showing hematopoietic cells (CD45+) and stromal cells (CD45−); (<b>e</b>) the proportion of stromal cells (CD45−), n = 5, *—<span class="html-italic">p</span> &lt; 0.05; (<b>f</b>) the proportion of hematopoietic cells (CD45+), n = 5, *—<span class="html-italic">p</span> &lt; 0.05. The data are presented as mean ± standard deviation (M ± SD); int (intact medium)—complete culture medium; ost—osteogenic culture medium; 1 g—standard culture conditions; smg—3D clinorotation for 14 days.</p> Full article ">Figure 4
<p>Histochemical evaluation of alkaline phosphatase activity in intact and osteoinduced heterotypic cell culture from murine bone marrow under 1 g and smg conditions. (<b>a</b>) Representative images, bright field microscopy, scale bar—100 μm. op 4 images are intact cells, and bottom 4 images are osteoinduced cells; (<b>b</b>) alkaline phosphatase activity assay by digital image processing, mean intensity, arb.un., n = 5, **—<span class="html-italic">p</span> &lt; 0.01. The data are presented as mean ± standard deviation (M ± SD). Abbreviations: int (intact medium)—complete culture medium; ost—osteogenic culture medium; 1 g—standard culture conditions; smg—3D clinorotation for 14 days.</p> Full article ">Figure 5
<p>Mineralized matrix in intact and osteoinduced heterotypic cell culture from murine bone marrow under 1 g and smg conditions, alizarin red S staining. (<b>a</b>) Representative images, bright field microscopy, scale bar—1000 μm (top row), 100 μm (bottom row). Top 4 images are intact cells, and bottom 4 images are osteoinduced cells; (<b>b</b>) quantitative analysis of alizarin red S staining intensity by digital image processing, mean intensity, arb.un., n = 5, **—<span class="html-italic">p</span> &lt; 0.01, ns - not significant. The data are presented as mean ± standard deviation (M ± SD). Abbreviations: int (intact medium)—complete culture medium; ost—osteogenic culture medium; 1 g—standard culture conditions; smg—3D clinorotation for 14 days.</p> Full article ">Figure 6
<p>Simulated microgravity effects on transcriptomic profile of bone marrow cell culture. (<b>a</b>,<b>b</b>) Differential gene expression of Hippo signaling pathway components and regulators (<span class="html-italic">Ajuba</span>, <span class="html-italic">Amot</span>, <span class="html-italic">Casp3</span>, <span class="html-italic">Dchs1</span>, <span class="html-italic">Fat4</span>, <span class="html-italic">Limd1</span>, <span class="html-italic">Lpp</span>, <span class="html-italic">Nf2</span>, <span class="html-italic">Wtip</span>, <span class="html-italic">Lats2</span>, <span class="html-italic">Mob1a</span>, <span class="html-italic">Mob1b</span>, <span class="html-italic">Sav1</span>, <span class="html-italic">Stk3</span>, <span class="html-italic">Meis1</span>, <span class="html-italic">Ptprz1</span>, <span class="html-italic">Lats1</span>, <span class="html-italic">Patj</span>, <span class="html-italic">Ywhaq</span>), transcription factors Taz and Yap1 (<span class="html-italic">Wwtr1</span>, <span class="html-italic">Yap1</span>), their mediator Tead2 (<span class="html-italic">Tead2</span>) and Runx2 (<span class="html-italic">Runx2</span>), as well as target genes of the transcription factors Yap/Taz (<span class="html-italic">Ccn2</span>, <span class="html-italic">Itgb2</span>) and Runx2 (<span class="html-italic">Alpl</span>). (<b>b</b>) Heatmap, n = 3, *—<span class="html-italic">p</span> &lt; 0.05. The data are presented as mean fold changes, smg vs. 1 g. Red—upregulation; blue—downregulation; white—no change; and gray—not assessed. Abbreviations: int (intact medium)—complete culture medium; ost—osteogenic culture medium; 1 g—standard culture conditions; smg—3D clinorotation for 14 days.</p> Full article ">Figure 7
<p>Confocal microscopy images of Yap1 in the intact and osteoinduced heterotypic cell cultures from murine bone marrow under 1 g and smg conditions. Representative confocal images of DAPI (blue)-, Yap1 (green)- and CD45 (red)-stained cell cultures. Scale bar—50 μm. Abbreviations: int (intact medium)—complete culture medium; ost—osteogenic culture medium; 1 g—standard culture conditions; smg—3D clinorotation for 14 days.</p> Full article ">Figure 8
<p>Yap1 translocation analysis in the intact and osteoinduced heterotypic cell cultures from murine bone marrow under 1 g and smg conditions. Quantitative analysis of confocal images. (<b>a</b>), (<b>b</b>)—The ratio of the mean fluorescence intensity (MFI) of nuclear and cytoplasmic Yap1 in the intact stromal cells (CD45−) and hematopoietic cells (CD45+), respectively. (<b>c</b>,<b>d</b>)—The ratio of the mean fluorescence intensity (MFI) of nuclear and cytoplasmic Yap1 in the osteoinduced cell culture containing stromal cells (CD45−) and hematopoietic cells (CD45+), respectively. The data from three independent experiments are presented as median ± interquartile range. ****—<span class="html-italic">p</span> &lt; 0.0001, ns - not significant. Abbreviations: int (intact medium)—complete culture medium; ost—osteogenic culture medium; 1 g—standard culture conditions; smg—3D clinorotation for 14 days.</p> Full article ">Figure 9
<p>Confocal microscopy of Runx2 in the intact and osteoinduced heterotypic cell cultures from murine bone marrow under 1 g and smg conditions. Representative confocal images of DAPI (blue)-, Runx2 (green)- and CD45 (red)-stained cell cultures. Scale bar—50 μm. Abbreviations: int (intact medium)—complete culture medium; ost—osteogenic culture medium; 1 g—standard culture conditions; smg—3D clinorotation for 14 days.</p> Full article ">Figure 10
<p>Analysis of Runx2 translocation in the intact and osteoinduced heterotypic cell cultures from mouse bone marrow under 1 g and smg conditions. Quantitative analysis of confocal images. (<b>a</b>,<b>b</b>)—The ratio of the mean fluorescence intensity (MFI) of nuclear and cytoplasmic Runx2 in the intact stromal cells (CD45−) and hematopoietic cells (CD45+), respectively. (<b>c</b>,<b>d</b>)—The ratio of the mean fluorescence intensity (MFI) of nuclear and cytoplasmic Runx2 in the osteoinduced cell culture containing stromal cells (CD45−) and hematopoietic cells (CD45+), respectively. The data from three independent experiments are presented as median ± interquartile range. ****—<span class="html-italic">p</span> &lt; 0.0001. Abbreviations: int (intact medium)—complete culture medium; ost—osteogenic culture medium; 1 g—standard culture conditions; smg—3D clinorotation for 14 days.</p> Full article ">

Figure 1
<p>Differentially expressed gene (DEG) profile of protein-coding and noncoding genes. DEG analysis of TNBC vs other subtypes with fold change filter of ≥1.5 and ≤ −1.5 FC was performed. Unsupervised clustering clearly showed two clusters. (<b>A</b>) Heat map shows all the variations including protein-coding and noncoding genes between TNBC and all other subtypes (Luminal A, Luminal B, Her2). (<b>B</b>) Heat map shows differential expression profile of lncRNA segregated through ENSEMBL-Biomart.</p> Full article ">Figure 2
<p>Comprehensive analysis of LUCAT1 in TNBC. (<b>A</b>) The list of DElncRNA from GSE192341 was compared to the lnc2cancer3.0 database, which is a collection of experimentally validated mechanistic, functional, and clinically relevant lncRNAs. Venn diagram shows comparative result with GSE192341 and only LUCAT1 was common in all the 3 categories (mechanism, function, and clinical). (<b>B</b>) LUCAT1 expression in TNBC and non-TNBC samples. Outliers or extreme values are shown as * in the figure. (<b>C</b>) ceRNA network of LUCAT1 using lncACTdb database. (<b>D</b>) GSEA of hallmark in cancer (C1; MSigDb) and oncogene signature pathways (C6; MSigDb) with LUCAT1-correlated genes (r² ≥ 0.3) show a significant enrichment in hallmark apical junction pathway (NES:1.57), complement pathway (NES: 1.48), TNF signaling via NFKB (NES: 1.46), oncogene IL2 (NES:1.49), and MEK (1.44) upregulation signatures.</p> Full article ">Figure 3
<p>LUCAT1 induces growth, proliferation and migration of TNBC cells. (<b>A</b>) Bar graph shows the expression profile of LUCAT1-associated miRNA (red bars) and mRNA (blue bars) in CCLE data of multiple breast cancer cell lines (TNBC vs. all other subtypes). (<b>B</b>) Bar graph shows the real-time mRNA expression profile of LUCAT1 in breast cancer subtype-specific cell lines. (<b>C</b>) TNBC cells were silenced for LUCAT1. RT-PCR results show mRNA expression level of LUCAT1 and GAPDH in TNBC cell lines. (<b>D</b>) MDA-MB-468 cells were silenced for LUCAT1 expression using siLUCAT1. The cell viability was evaluated using MTT assay and the data presented as % viability showing the mean viability ± SD of 6 independent replicates. (<b>E</b>) Trypan blue live–dead staining of MDA-MB-468 cells transfected with scramble-control and LUCAT1 siRNA was conducted. The bar graph shows the number of live cells only. *** <span class="html-italic">p</span> = 1.65046 × 10⁻⁶. (<b>F</b>) TNBC cells were silenced for LUCAT1 expression. Representative pictures of colony-formation assay in TNBC cells in the presence and absence of LUCAT1 are shown. (<b>G</b>) TNBC cells were silenced for LUCAT1 expression and subjected to transwell migration assay. Representative images show the cells migrated through the upper chamber in scramble-control and LUCAT1 siRNA group. Images captured at 10× magnification.</p> Full article ">Figure 4
<p>LUCAT1 functions as an important axis regulating drug efflux, stemness, and programmed cell death in TNBC cells. (<b>A</b>) TNBC cells were silenced for LUCAT1. Identification of side population (SP) cells from multiple TNBC cells. SP = side population. SP cells of HCC1806, HCC1937 and Hs578t were analyzed by dual-wavelength flow cytometer after staining with Hoechst 33342. (<b>B</b>) Hs578t cells were silenced for LUCAT1, and mRNA-expression level of stemness markers (OCT-4, CMYC, and ZEB1) were examined. (<b>C</b>) HCC1806 cells were silenced for LUCAT1, and CD49F level was examined using FACS analysis. (<b>D</b>) HCC1937 cells were silenced for LUCAT1 and ALDH activity assay was performed. (<b>E</b>) Total protein lysates from HCC1806 cells transfected with scramble-control and LUCAT1-siRNA were immunoblotted for the expression of BAX, Bcl2-xL, and T-PARP. (<b>F</b>) Flow cytometry analysis of cell distribution in respective phases of cell cycle of HCC1806 cells transfected with scramble-control and LUCAT1 siRNA. Sub-G1 cells correspond to apoptotic cells. (<b>G</b>) mRNA expression level of WNT1, WNT2, LUCAT1 and GAPDH in HCC1806 cells transfected with scramble-control and LUCAT1 siRNA. (<b>H</b>) TOPflash/FOPflash reporter assay in HCC1937 cells transfected with scramble-control and LUCAT1 siRNA (** <span class="html-italic">p</span> = 0.00252).</p> Full article ">Figure 5
<p>Association between LUCAT1 and miRNA, and LUCAT1 and mRNAs in TNBC. (<b>A</b>) Kaplan–Meier plots of triple-negative breast cancer survival in relation to high LUCAT1 + low miRs (miR-375 + miR-642a + miR-200c + miR-181-5p + miR-199a-5p + miR-493-5p + miR-181c-5p). (<b>B</b>) Kaplan–Meier plots of triple-negative breast cancer survival in relation to high LUCAT1 + high target genes (WNT + YAP + DNMT + ABACb + Zeb + LRP + ROCK + ADAM + ABCC).</p> Full article ">

Figure 1
<p>Workflow of the study. (<b>A</b>) PDAC cells were exposed to gemcitabine (GEM) or paclitaxel (PTX) to generate chemoresistant clones. (<b>B</b>) PDAC cells, and their resistant subclones, seeded on elastic micropillar arrays of varying stiffness were assessed for force generation by measuring the pillar deflections. Traction forces were defined as the inward-pointing forces. (<b>C</b>) Single-cell motility was assessed in cells seeded on collagen- and fibronectin-coated substrates. (<b>D</b>) The 3D collagen-embedded spheroid invasion and spheroid-induced ECM remodeling were analyzed. (<b>E</b>) YAP nuclear translocation assessed by immunofluorescence for cells growing on soft pillars. (<b>F</b>) Biomarkers of epithelial-to-mesenchymal transition (EMT) were assessed by RT-qPCR. Part of the figure was adapted from images made by Servier Medical Art by Servier, licensed under a Creative Commons Attribution 4.0 Unported License, at <a href="https://smart.servier.com" target="_blank">https://smart.servier.com</a>.</p> Full article ">Figure 2
<p>PDAC cell spreading area varies with stiffness. (<b>A</b>) Representative confocal microscopy images of BxPC-3 cells (green) growing on fibronectin-coated pillars (red) of different stiffness. Scale bar: 10 µm (<b>B</b>) Boxplots of cell spreading area (μm²) for BxPC-3, CAPAN-1, HPDE, SUIT-2.028, and SUIT-2.007 growing on fibronectin-coated pillars (25th and 75th percentiles marked, line at median).</p> Full article ">Figure 3
<p>PDAC cell traction forces increase with substrate stiffness, but not with metastatic potential. (<b>A</b>) Representative linear regression of the total forces (nN) vs. spreading area (μm²) of SUIT-2.028. All the linear regression models of the other cell lines are found in <a href="#app1-cancers-16-03863" class="html-app">Supplemental Figure S1</a>. In all the measurements, the regression coefficient was R² &gt; 0.45. (<b>B</b>) Representative confocal microscopy images of the traction forces of the BxPC-3 cells (green) growing on fibronectin-coated pillars (red). White arrows indicate cellular traction forces on the pillars. (<b>C</b>) Boxplots of the PDAC cell traction forces expressed as mean force per pillar (nN) (25th and 75th percentiles marked, line at median). Statistical significance was calculated using the softest condition (11 kPa) as the reference group. (<b>D</b>) Mean force per pillar (nN) of the PDAC cell lines of different phenotypes. The results from other stiffness values (11, 29, and 142 kPa) are shown in <a href="#app1-cancers-16-03863" class="html-app">Supplemental Figure S3</a> and pillar‘s background forces are shown in <a href="#app1-cancers-16-03863" class="html-app">Supplemental Figure S2</a>. Each dot of plots in (<b>A</b>,<b>C</b>,<b>D</b>) represents the result from one cell. (<b>C</b>,<b>D</b>) Statistical significance was set at <span class="html-italic">p</span> &lt; 0.05 and is indicated by ****, <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 4
<p>Chemoresistant PDAC cells apply higher traction forces. (<b>A</b>) Representative confocal microscopy images of the traction forces of SUIT-2.028 WT, GR, and PR cells growing on fibronectin-coated soft (11 kPa) and stiff (47 kPa) pillars. White arrows indicate traction forces that the cells applied to deflect the pillars. Nucleus is indicated by cyan color (DAPI) and cytoskeleton by green color (AlexaFluor568 Phalloidin). (<b>B</b>) Boxplots of PDAC cell traction forces on the soft and stiff pillars on the left and right, respectively, expressed as mean force per pillar (nN) (25th and 75th percentiles marked, and line at median). Statistical significance was calculated using the parental cells (WT) as the reference group. Each dot represents one cell analyzed. Statistical significance was set at <span class="html-italic">p</span> &lt; 0.05 and is indicated by *, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01; ***, <span class="html-italic">p</span> &lt; 0.001; ****, <span class="html-italic">p</span> &lt; 0.0001, “ns” means not significant.</p> Full article ">Figure 5
<p>PDAC resistant single-cell migration is different from parental cells. PDAC single-cell mean velocity (μm/min), growing on (<b>A</b>) collagen-coated or (<b>D</b>) fibronectin-coated wells. The directionality of PDAC cell migration trajectories growing on collagen-coated wells, expressed as (<b>B</b>) diffusive fraction = DF and (<b>C</b>) diffusion constant. The directionality of PDAC cell migration trajectories growing on fibronectin-coated wells, expressed as (<b>E</b>) DF fraction and (<b>F</b>) diffusion constant. All the conditions are represented as boxplots with the smallest and largest values marked, and line at median. The statistical significance was calculated using the parental cells (WT) as the reference group. Each dot represents the population mean for one section of the well. (<b>A</b>–<b>F</b>) Statistical significance was set at <span class="html-italic">p</span> &lt; 0.05 and is indicated by *, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01; ***, <span class="html-italic">p</span> &lt; 0.001; ****, <span class="html-italic">p</span> &lt; 0.0001, “ns” means not significant.</p> Full article ">Figure 6
<p>PDAC chemoresistant cell ability for 3D ECM remodeling and invasion. (<b>A</b>) Representative collagen fibers alignment (left columns), obtained by reflection microscopy, and actin (right columns) of PDAC chemoresistant spheroids. Scale bar: 200 µm (<b>B</b>) Polar plots representing the percentage of the frequency of the distribution of collagen fibers angles from 0° to 90°. Each bar represents the % of fibers in a sector of 5 degrees, expressed as the mean of 2 biological replicates, with at least 4 technical replicates. Lines of the darker shade of the bars represent the upper and lower bounds of SD of the % of collagen fibers in each sector. Orange lines represent the mean % of the frequency of the respective WT for each cell line to facilitate the comparison. (<b>C</b>) Relative area covered by spheroids after 2 days. Dots represent the value of individual spheroids. Data are expressed as mean ± SD. (<b>D</b>) Percentage of aligned fibers, defined as fibers comprised between the angles of 72.5 and 90, which means that those fibers are perpendicular to the closest point of the spheroid. Dots represent the value of individual spheroids. (<b>C</b>,<b>D</b>) Statistical significance was set at <span class="html-italic">p</span> &lt; 0.05 and is indicated by ****, <span class="html-italic">p</span> &lt; 0.0001. “ns” means not significant.</p> Full article ">Figure 7
<p>PDAC chemoresistant cell differential mechanobiology does not rely on YAP nuclear translocation nor on EMT switch. (<b>A</b>) Relative gene expression of <span class="html-italic">E-cadherin</span>, <span class="html-italic">N-cadherin</span>, and <span class="html-italic">Vimentin</span> as assessed by RT-qPCR. Data are expressed as the mean ± SD of three independent experiments. (<b>B</b>) Representative confocal microscopy images of SUIT-2.028 cells growing on soft (11 kPa) pillars and stained with YAP. Scale bar: 10 µm (<b>C</b>) YAP nuclear translocation, expressed as % of nuclear YAP over total YAP in SUIT-2.028 and SUIT-2.007. (<b>D</b>) Linear regression model between the mean force per pillar vs. % of nuclear YAP in SUIT-2.007 (left panel) and SUIT-2.028 (right panel). Each dot in (<b>C</b>,<b>D</b>) represents one cell. *, <span class="html-italic">p</span> &lt; 0.05; **, <span class="html-italic">p</span> &lt; 0.01; ***, <span class="html-italic">p</span> &lt; 0.001; ****, <span class="html-italic">p</span> &lt; 0.0001, “ns” means not significant.</p> Full article ">Figure 8
<p>PDAC resistance leads to changes in the mechanobiological signatures of cells, the detailed characteristics of which yet depend on cell type and ECM dimensionality. Part of the figure was adapted from images made by Servier Medical Art by Servier, licensed under a Creative Commons Attribution 4.0 Unported License, at <a href="https://smart.servier.com" target="_blank">https://smart.servier.com</a>.</p> Full article ">

Figure 1
<p>Mechanical properties of adipose tissue and physical characteristics of hydrogels at different concentrations. (<b>A</b>) Results of stress and elastic modulus tests on different regions of adipose tissue. (<b>B</b>) Statistical results of stress testing in adipose tissue. (<b>C</b>) Testing results of the elastic modulus in various regions of adipose tissue (n = 5). (<b>D</b>) Stress testing results of hydrogels at different concentrations. (<b>E</b>) Elastic modulus testing results of hydrogels at varying concentrations (n = 5). (<b>F</b>) SEM images of hydrogels at different concentrations. (<b>G</b>) Statistical analysis of pore sizes in hydrogels at different concentrations (n = 80). The pore diameters were analyzed using Nano Measurer 1.2 software and statistically processed using GraphPad Prism 8.0.2 software.* <span class="html-italic">p</span> &lt; 0.05. **** <span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 2
<p>Comprehensive assessment of the effects of hydrogels on adipocyte function and material mechanical properties. (<b>A</b>) Observation of adipocyte differentiation in hydrogels via fluorescence staining. (<b>B</b>) Quantitative analysis of fluorescence signals related to adipocyte differentiation. (<b>C</b>) Measurement of the expression levels of specific genes associated with adipocyte differentiation. (<b>D</b>) Evaluation of adipocyte viability within the hydrogels. (<b>E</b>) Assessment of the impact of cell embedding on hydrogel contraction (n = 5). (<b>F</b>) Monitoring of cell proliferation in hydrogels over different culture time periods. (<b>G</b>) Statistical analysis of cell viability data. Fluorescence intensity was analyzed using ImageJ 1.52P software and statistically processed using GraphPad Prism 8.0.2 software. * <span class="html-italic">p</span> &lt; 0.05. ** <span class="html-italic">p</span> &lt; 0.01. *** <span class="html-italic">p</span> &lt; 0.001. **** <span class="html-italic">p</span> &lt; 0.0001. ns: not significant (n = 4).</p> Full article ">Figure 3
<p>Immunofluorescence co-localization and protein analysis of YAP expression in hydrogels. (<b>A</b>) Immunofluorescence staining of YAP in adipocytes cultured in hydrogels of different concentrations. YAP is shown in green, and the nucleus is stained with DAPI (blue), with the merged images displaying co-localization. In the histogram, the red represents YAP signals (green fluorescence channel), while the blue represents DAPI signals (nuclei). (<b>B</b>) A single cell is outlined as the test subject, as shown in (<b>A</b>). The nucleus is labeled in blue and YAP is labeled in green, outlined by a white line. The fluorescence intensity distribution of YAP and DAPI along the white box is analyzed, with the intensity values normalized to the average YAP intensity. (<b>C</b>) Percentage of YAP co-localized with the nucleus. (<b>D</b>) Proportion of YAP signals not overlapping with the nucleus. (<b>E</b>) Percentage of YAP localized within the nucleus. (<b>F</b>) Western blot analysis of YAP protein expression in varying hydrogel concentrations. (<b>G</b>) Quantitative analysis of YAP protein expression across varying hydrogel concentrations. (<b>H</b>) Western blot analysis of phosphorylated YAP (P-YAP) protein expression in hydrogels of different concentrations. (<b>I</b>) Quantitative analysis of P-YAP protein expression across various hydrogel concentrations was conducted. Fluorescence intensity and co-localization data were processed with ImageJ 1.52P software, and statistical analysis was executed using GraphPad Prism 8.0.2 software. Statistical significance is indicated as * <span class="html-italic">p</span> &lt; 0.05. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, and ns for not significant (n = 3).</p> Full article ">Figure 4
<p>Inhibition of the YAP signaling pathway promotes adipogenesis. (<b>A</b>) BODIPY fluorescence staining of lipid droplets following inhibition of the YAP signaling pathway using the VP inhibitor. (<b>B</b>) Fluorescence intensity analysis of lipid droplets in hydrogels of different concentrations. (<b>C</b>) Changes in YAP/P-YAP protein levels after VP inhibition. (<b>D</b>,<b>E</b>) Quantitative analysis of protein expression changes after YAP signaling inhibition. The fluorescence intensity in Figures (<b>A</b>) and (<b>C</b>) was analyzed using ImageJ 1.52P software, and statistical data were processed using GraphPad Prism 8.0.2 software. Statistical significance is denoted as ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, **** <span class="html-italic">p</span> &lt; 0.0001, and ns for non-significant differences (n = 3).</p> Full article ">Figure 5
<p>Effect of hydrogel stiffness on adipocyte migration. (<b>A</b>) Cell staining at the bottom of the 8 µm pore size chamber, showing adipocyte migration characteristics in hydrogels of different concentrations. (<b>B</b>) Schematic diagram of the co-culture system featuring an 8 µm pore size upper chamber and a lower 24-well plate for observing cell migration behavior. (<b>C</b>) Cell migration in the bottom of the 24-well plate, with lipid droplets stained green using BODIPY and actin filaments stained red using fluorescence staining. (<b>D</b>) Quantitative analysis of cell migration. Fluorescence intensity was analyzed using ImageJ 1.52P software and statistically processed using GraphPad Prism 8.0.2 software. **** <span class="html-italic">p</span> &lt; 0.0001. (n = 3).</p> Full article ">Figure 6
<p>Adipocyte differentiation is regulated by the YAP signaling pathway. (<b>A</b>,<b>B</b>) show collagen hydrogels of different stiffness. In softer hydrogels (<b>A</b>), the translocation of YAP from the nucleus to the cytoplasm is diminished, along with reduced phosphorylation, resulting in decreased lipid droplet accumulation. In contrast, in stiffer hydrogels (<b>B</b>), YAP translocates more readily from the cytoplasm to the nucleus, accompanied by elevated phosphorylation, which enhances lipid droplet accumulation. Hydrogels with varying matrix stiffness influence YAP’s relocalization between the nucleus and cytoplasm under mechanical stress. When YAP remains in the cytoplasm, it undergoes phosphorylation, promoting the initiation of the adipocyte differentiation program. Therefore, matrix stiffness ultimately affects adipocyte differentiation by regulating both the cellular localization and activity state of YAP. This figure was created with Figdraw (HOME for Researchers).</p> Full article ">