Forward genetic analysis of sleep in randomly mutagenized mice

Animals and mutagenesis

Male C57BL/6J mice (CLEA Japan) were treated with ethylnitrosourea (85 mg/kg, Sigma-Aldrich) by intraperitoneal injection twice at weekly intervals at the age of 8 weeks. At the age of 25-30 weeks, the sperm of the mice was used for in vitro fertilization with eggs of C57BL/6N mice to obtain F1 offspring. Mice were provided food and water ad libitum, and were maintained on a 12-hour light/dark cycle and housed under controlled temperature and humidity conditions. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Tsukuba and the RIKEN BioResource Center, University of Texas Southwestern Medical Center at Dallas.

Surgery

EEG/EMG electrode implantation was performed as described previously³⁴, with isoflurane (3% for induction, 1% for maintenance) used for anesthesia. Seven days after surgery, the mice were tethered to a counterbalanced arm (Instech Laboratories) that allowed free movement and exerted minimal weight.

Screening scheme

At the age of 12 weeks, male mice were implanted with EEG/EMG electrodes and then screened for sleep/wakefulness behavior. Examined parameters were total time spent in wake, NREMS and REMS, episode duration of wake, NREMS and REMS, appearance of muscle atonia during REMS, and rebound sleep after 4-h sleep deprivation by shaking the cages. For quantitative parameters, we selected mice whose phenotypes deviated from the average by at least 3 standard deviations. After confirming the reproducibility of the sleep phenotype, the mice were selected for offspring production by natural mating or IVF with wildtype females to examine the heritability of the sleep phenotypes. If at least 30% of the male littermates showed sleep phenotypes similar to their father, we considered the sleep abnormalities to be heritable.

Linkage analysis

SNPs of N2 mice were determined using a custom TaqMan Genotyping assay (Thermo Fisher). The custom probes were designed based on the polymorphism data between C57BL/6J and C57BL/6N²⁰. QTL analysis was performed using J/qtl software (Jackson Laboratory).

Whole exome sequencing

Whole exomes were captured with SureSelectXT2 Mouse All Exon (Agilent) and processed to a paired end 2 × 100-bp run on the Illumina HiSeq2000 platform at the UTSW McDermott Center Next Generation Sequencing Core. Reads were mapped to the University of California Santa Cruz mm9 genome reference sequence for C57BL/6J using Burrows-Wheeler Aligner and quality filtered using SAMtools. Cleaned BAM files were used to realign data and call variants using the Genome Analysis ToolKit to detect heterozygous mutations.

Sleep behavior analysis

The recording room was kept under 12-h light-dark cycles and a constant temperature (24-25 °C). To examine sleep-wake behavior under baseline conditions, EEG/EMG signals were recorded for two consecutive days from the onset of the light phase. EEG/EMG data were visualized and analyzed using a MatLab (MathWorks)-based, custom semi-automated staging program followed by visual inspection. EEG signals were subjected to fast Fourier transform analysis from 1 to 30 Hz with 1-Hz bin using MatLab-based custom software. Epochs containing movement artifacts were included in the state totals but excluded from subsequent spectral analysis. Sleep/wakefulness was staged into wakefulness, NREM sleep and REM sleep. Wakefulness was scored based on the presence of low amplitude, fast EEG and high amplitude, variable EMG.

NREMS was characterized by high amplitude, delta (1-4 Hz) frequency EEG and low EMG tonus, whereas REMS was staged based on theta (6-9 Hz) dominant EEG and EMG atonia. Hourly delta density during NREMS indicates hourly averages of delta density which is the ratio of delta power to total EEG power at each 20-second epoch. For the power spectrum of sleep/wakefulness, EEG power of each frequency bins was expressed as a percentage of the total EEG power over all frequency bins (1-30Hz) and sleep/wakefulness states^34,35. For sleep deprivation, mice were sleep deprived for 2, 4 and 6 hours from the onset of the light phase by gently touching the cages when they started to recline and lower their heads. Food and water were available. To evaluate the effect of sleep deprivation, NREM delta power during the first hour after sleep deprivation was expressed relative to the same zeitgeber time of the basal recording or relative to the mean of the basal recording. For caffeine and modafinil injection experiments, mice were fully acclimatized for intraperitoneal injection before sleep recording. After 24-h baseline recording, mice received intraperitoneally caffeine (Sigma), modafinil (Sigma) or vehicle (0.5% methyl cellulose (Wako)) at ZT0, followed by 12-h recording. Injections were delivered once per week, with each injection followed by a 6-8 day washout period, during which mice remained in the recording chamber. To examine the sleep/wakefulness behavior under constant darkness, after 48-h recording under a LD 12: 12 cycle, EEG/EMG recording continued in constant darkness for 3 days.

Circadian behavior analysis

Mice were housed individually in a cage (Width 23 cm, Length 33 cm, Height 14 cm) containing a wireless running wheel (Med Associate #ENV-044). Cages were placed in a light-tight chamber equipped with green LED light (100 lux at the bottom of the cage). The rotation numbers of wheels were obtained with 1-min bin using Wheel manager software (Med Associate). Mice were entrained to LD12:12 cycle for 7 days, and then released into constant darkness for 3 weeks. The free running period was calculated with linear regression analysis of activity onset using MatLab-based custom software. Circadian activity amplitude was calculated by fast Fourier transform of activity data which were processed with Bartlett window using MatLab-based custom software. Relative amplitude was normalized to the mean amplitude of the wild type group.

Western blot

A rabbit polyclonal antibody against the C-terminal 171 amino acids of mouse SIK3 was generated using custom antibody production service (Pacific Immunology, Ramona, CA USA). Tissues were homogenized using a rotor-stator homogenizer (Polytron) in ice-cold lysis buffer (20 mM HEPES pH7.5, 100 mM NaCl, 10 mM Na4P2O7, 1.5% Triton-X100,15 mM NaF, 1X PhosSTOP (Roche), 5 mM EDTA, 1X Protease Inhibitor (Roche)), and then centrifuged at 13,000g at 4 °C. The supernatants were separated by SDS-PAGE and transferred on PVDF membrane. Western blotting was performed according to standard protocols.

In situ hybridization

In situ hybridization was performed as described previously³⁶. In brief, a 0.7-0.8 kb fragment of Nalcn cDNA was inserted into pGEM-T easy (Promega) and used for DIG-labeled probe synthesis. Mice were deeply anesthetized with sodium pentobarbital and perfused transcardially with PBS followed by 4% paraformaldehyde (PFA). Forty μm-thick brain sections were treated with 0.3% Triton X-100, digested with 1 μg/ml proteinase K, treated with 0.75% glycine, and then treated with 0.25% acetic anhydride in 0.1 M triethanolamine. After overnight incubation with DIG-labeled probe at 60 °C, the sections were washed and then incubated with alkaline phosphatase-conjugated anti-DIG Fab fragments (Roche, 11175041910). The reactions were visualized with a 5-bromo-4-chloro-3-indolyl-phosphate/4-nitroblue tetrazolium (BCIP/ NBT) substrate solution (Roche).

Cell lines

HEK293 cells (RCB1637) and HEK293T cells (RCB2202) were obtained from the RIKEN BRC Cell Bank. Cells were cultured in DMEM (Wako) supplemented with 10% FBS, 1% GlutaMAX (Thermo Fisher Scientific), and penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO₂. Cell lines were regularly tested for mycoplasma contamination using MycoAlert (Lonza). Cell lines were regularly renewed by obtaining cell stocks from the Cell Bank for authentication. We used HEK293 and HEK293T cells because of their reliable growth, high efficiency in transfection and morphology suitable for electrophysiological experiments.

Production of Sik3 gene-modified mice by conventional gene targeting

For generating Sik3^Slp knock-in mice, a genomic fragment containing exon 13 of the Sik3 gene was isolated from C57BL/6 mouse genomic BAC clone from a RP23 mouse genomic BAC library (Advanced GenoTEchs Co). A 1.7-kb fragment of FRT-PGK-gb2-neo-FRT-loxP cassette (Gene Bridges) flanked by two flippase recognition target (FRT) sites was inserted before exon 12. The targeting vector also contains a G-to-A substitution at the 5th nucleotide from the beginning of intron 13. The targeting vector was linearized and electroporated into the C57BL/6N ES cell line RENKA. Correctly targeted clones were injected into eight-cell stage ICR mouse embryos, which were cultured to produce blastocysts and then transferred to pseudopregnant ICR females. Resulting male chimeric mice were crossed with female C57BL/6N mice to establish the Sik3^Slp-neo/+ line. To remove the neomycin resistance gene with the FLP-FRT system, Sik3^Slp-neo/+ mice were crossed with Actb-FLP knock-in mice.

Production of Sik3 gene-modified mice using ZFN

The custom designed ZFN mRNAs targeting the exon 13-intron 13 boundary region of the Sik3 gene were obtained from Sigma-Aldrich’s Composers® Custom ZFN service. Before the final assembly of the ZFN products, Sigma-Aldrich validated the designed ZFN binding sequences in silico using their bioinformatics tools and in vitro using Nero2A cell-lines, ensuring high cutting efficiency and specificity using mismatch-specific endonuclease CelI according to the manufacturer’s instructions. The ZFN mRNAs were injected into single-cell stage C57BL/6J mouse zygotes at UT Southwestern Transgenic Core facility. The injected eggs were then transferred to pseudopregnant females to generate F0 founders. In total, 45 out of 96 F0 mice were found to be modified at the exon 13-intron 13 boundary region of the Sik3 gene. We crossed one F0 male mouse which had a 2 bp deletion from the last nucleotide of exon 13 with female C57BL/6N mice to obtain F1 mice of Sik3^Slp/+ ZFN. The F1 mice were used to confirm the skipping of exon13 in Sik3 mRNA which was purified from the brains and livers. The F2 male mice were used for sleep/wakefulness behavior analysis.

Production of Flag-Sik3 mice and Nalcn^DRL mice by CRISPR/Cas9 technology

To produce a Cas9/single guide RNA (sgRNA) expression vector, oligo DNAs (5’-caccGCGAGCGGCCATCGACCCGC-3’ and 5’-aaacGCGGGTCGATGGCCGCTCGC-3’) were annealed and then inserted into pX330 vector (Addgene). The cleavage activity of the pX330-Sik3Ex1 vector was evaluated by the EGxxFP system³⁷. Genomic DNA containing exon 1 of the Sik3 gene was amplified and inserted into pCAG-EGxxFP to produce pCAG-EGxxFP-Sik3Ex1. The pX330-Sik3Ex1 and pCAG-EGxxFP-Sik3Ex1 were transfected into HEK293 cells. As a donor oligonucleotide, a single-stranded 200nt DNA was synthesized (Integrated DNA Technologies), which contained a FLAG-HA-coding sequence in the center and 70nt arms at the 5’ and 3’ ends. Female C57BL/6J mice or Sik3^Slp knock-in mice were injected with pregnant mare serum gonadotropin and human chorionic gonadotropin at a 48-h interval, and mated with male C57BL/6J mice. The fertilized one-cell embryos were collected from the oviducts. Then, 5 ng/μl of pX330-Sik3Ex1 vector and 10 ng/μl of the donor oligo were injected into the pronuclei of these one-cell-stage embryos. The injected one-cell embryos were then transferred into pseudopregnant ICR mice. F0 mice were genotyped for the presence of FLAG-coding sequence in exon1 of the Sik3 gene and for the presence of the Sik3^Slp mutation. F0 mice containing FLAG-SIK3 were further examined for the presence of the Cas9 transgene and off-target effects. Candidate off-target sites were identified based on a complete match of 16 bp at the 3’ end, including the PAM sequence. F0 mice were mated with C57BL/6N mice to obtain F1 offspring.

Nalcn^Drl mice were produced as described above. To produce the sgRNA expression vector, pX330-NalcnEx9, oligo DNAs (5’-caccAGCAATAAACACATTCTGAA-3’ and 5’-aaacTTCAGAATGTGTTTATTGCT-3’) were used. Genomic DNA containing exon 9 of the Nalcn gene was amplified and inserted into pCAG-EGxxFP to produce pCAG-EGxxFP-NalcnEx9. As a donor oligo, a single-stranded 199nt DNA containing a T-to-A substitution at the center was synthesized (Integrated DNA Technologies). Nalcn mutant mice of N2-N3 generation were used for sleep/wakefulness analysis.

Phosphoproteomic analysis

To evaluate FLAG-tagged SIK3 protein in brains, we performed peptide mapping of the purified FLAG-SIK3 protein. The brains of Flag-Sik3 knock-in mice and Flag-Sik3^Slp knock-in were quickly dissected after cervical dislocation. Brains were homogenized in detergent-free buffer and then centrifuged (100,000 × g, 30 min, 4 °C ). The supernatant was immunoprecipitated with anti-DDDDK antibody beads (MBL #3325). The eluate was run on a polyacrylamide ge1 and stained with SilverQuest Silver staining kit (Life technologies). FLAG-SIK3 band (150kD) was dissected with a fresh blade. The proteins in the bands were reduced with lOmM dithiothreitol and alkylated with 40 mM iodoacetamide. Each sample was digested with trypsin (4 μg/ml; Trypsin Gold, Promega) at 37 °C overnight. The extracted peptides were then separated via nano flow LC (Advance LC, Michrom Bioresources) using a C18 column. The LC eluate was coupled to a nano-ionspray source attached to a Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific). All MS/MS spectra were searched using Proteome Discoverer 1.3 software (Thermo Fisher Scientific). Peptides were mapped through mouse SIK3 (NP_081774) with 56% coverage.

To examine the effect of sleep deprivation on the phosphorylation status of SIK3 protein, Five Flag-Sik3 knock-in mice or five Flag-Sik3^Slp knock-in mice were ad lib slept (S) or sleep deprived (SD) for four hours by gentle handling immediately after light onset (ZT0-ZT4). Five wildtype (WT) mice were used as a negative control. At ZT4, mouse brains were quickly dissected after cervical dislocation, rinsed with cold PBS, and snap frozen in liquid nitrogen. Each half of the brains was lysed in 2 ml of ice-cold lysis buffer (20 mM HEPES (pH7.4), 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 2 mM MgCl₂, 15 mM NaF, 10 mM Na₄P₂O₇) freshly supplemented with protease/phosphatase inhibitor cocktail tablets (Roche), and homogenized in a glass tissue homogenizer. After brain homogenate was incubated for 30 min and centrifuged at 13,000 g for 20 min at 4 °C, the supernatant was pre-cleared by IgG and Protein G beads for 30 min before immunoprecipitation. Each pre-cleared lysate was added to 50 μl of anti-FLAG antibody-conjugated Sepharose beads (Sigma, A2220) and rotated overnight at 4 °C. After washing the beads 5 times with cold wash buffer (20 mM HEPES (pH7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM MgCl₂, 15 mM NaF, 10 mMNa4P₂0₇), 50μl of elution buffer (2%SDS, 60 mM Tris/HCl (pH6.8), 50 mM DTT, 10% glycerol) was added and rotated for 10 min at 4 °C. Elution was repeated twice and combined into one eluate and analyzed by western blotting. For each group of five mice, the five eluates of were mixed and equally split into two samples for mass spectrometric analysis. Thus, a total of six samples were reduced, alkylated, and trypsin digested overnight. After each sample was labeled with a different TMT-6 reagent (Thermo Fisher Scientific), six samples were combined into one mixture for HPLC fractionation using a Cl8 column. A total of 12 fractions were collected, and analyzed separately on the Orbitrap-Fusion mass spectrometry platform (Thermo Fisher Scientific) using a reverse-phase LC-MS/MS method. We performed data analysis to identify peptides and quantified reporter ion relative abundance using Proteome Discoverer 2.1 (Thermo Fisher Scientific). The relative abundance of quantified SIK3 phosphorylation sites was normalized with wildtype negative control and total SIK3 protein abundance.

Patch-clamp recordings from HEK cells

To express wildtype NALCN, we used pTracer-CMV2-ratNALCN-EF1α-EGFP (a gift from Dr. Ren)³⁸. A single nucleotide substitution was induced to make pTracer-CMV2-ratNALCN(DRL)-EF1α-EGFP using a KOD plus Mutagenesis kit (Toyobo). HEK293T cells were grown to −50% confluency in 12-well plates. Using Lipofectamine LTX (2 μl) and PLUS (1 μl) reagent (Thermo Fisher Scientific), the cells were cotransfected with 0.3 μg of each plasmid DNA encoding rat NALCN-EGFP (WT or DRL), mouse UNC-80, and mouse SRC (Y529F) (constitutively active Src) in 12-well plates. UNC-80 and SRC kinase activate NALCN²⁷,²⁸. In some experiments, the cells were incubated with 10 μM Gd³⁺ to inhibit NALCN. The cells were dissociated and plated on 18-mm coverslips coated with poly-L-lysine in fresh culture medium prior to patch-clamp recordings.

All patch-clamp recordings from HEK293T cells were performed > 72 h after transfection. Recording patch pipettes were pulled from glass capillaries (1B150F-4, World Precision Instruments) using a micropipette puller (P-97, Sutter Instrument) to give a resistance of ~9 MΩ. The series resistance of whole-cell recordings was ~40 MΩ, which was not compensated. Patch pipettes were fdled with solution containing 150 mM CsOH, 120 mM methanesulfonic acid, 10 mM NaCl, 10 mM EGTA, 2 mM Mg₂ATP and 10 mM HEPES (pH 7.4 adjusted with methanesulfonic acid; osmolarity, 290–299 mOsm/L adjusted with CsC1). The cells on coverslips were transferred to a recording chamber under a fluorescence upright microscope (Axio Examiner D1, Zeiss) and continuously perfused with the bath solutions containing 150 mM NaCl, 3.5 mM KCl, 10 mM HEPES, 20 mM glucose, 5 mM NaOH, 2 mM MgCl₂ and 1.2 mM CaCl₂ (pH 7.4 adjusted; osmolarity, 300–310 mOsm/L). The transfected cells were identified by EGFP fluorescence. Patch-clamp recordings were performed at room temperature (24 °C) using a computer-controlled amplifier (MultiClamp 700B, Molecular Devices). The signals were digitized with A/D converter (Digidata 1440A, Molecular Devices), and acquired with Clampex (Molecular Devices) at a sampling rate of 50 kHz, and low-pass filtered at 5 kHz. At the end of recording, Gd³⁺ (10 μM) was used to confirm that the whole-cell currents were mediated through NALCN³⁸. Data were analyzed using Clampfit (Molecular Devices). The equilibrium potentials were calculated from I-V curves. Mean membrane conductance was estimated from the regression lines fitted to I-V curves from individual cells. Current, membrane conductance and charge transfer were normalized to membrane capacitance.

Patch-clamp recordings from neurons

Patch pipettes and recording system were the same as those used in recordings from HEK293 cells. Acute brain slices containing the DpMe were prepared from postnatal day 12-23 Nalcn^+/+ or Nalcn^Drl/+ mice. After the induction of deep anesthesia with isoflurane, mice were decapitated and the brains were rapidly removed into an ice-cold cutting solution containing 2.5 mM KCl, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 25 mM glucose, 185 mM sucrose, 0.5 mM CaCl₂ and 10 mM MgCl₂ (pH 7.4, when bubbled with 95% O₂ and 5% CO₂). The brains were cut coronally into 200-250 μm-thick slices with a vibratome (VT-1200S, Leica). The slices were incubated at 37 °C for 1 hr in artificial cerebrospinal fluid (aCSF) containing 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 10 mM glucose, 2 mM CaCl₂ and 1 mM MgCl₂ (pH 7.4, when bubbled with 95% O₂ and 5% CO₂) prior to recordings. Slices were transferred to a recording chamber perfused with aCSF under an upright microscope (Axio Examiner D1, Zeiss). For current-clamp recordings, patch pipettes were filled with solution containing 125 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 0.5 mM EGTA, 8 mM Phosphocreatine-Na₂, 4 ATP-Mg and 0.3 GTP-Na (pH 7.3 adjusted with KOH; osmolality, 290 mOsm/L). The DpMe was identified with axon bundles. Recordings were made from cells located in the medial part of the DpMe. Cells showing no action potentials following current injection (> 1 nA, > 5 ms) were discarded from analysis. Membrane potentials were recorded for 1–10 min.

Fruit fly stocks and behavioral assay

Sik3 hypomorph and UAS-Sik3, UAS-Sik3(S563A) transgenic flies were gifts from Drs. Marc Montminy and John B. Thomas³⁹. elav-GS (GeneSwitch) stocks were from the Bloomington stock center. Flies were reared at 25°C under 12 hr light : 12 hr dark cycle (LD) in 50–60% relative humidity on a standard fly food consisting of corn meal, yeast, glucose, wheat germ and agar.

Sleep analysis was performed as described previously⁴⁰. Briefly, male flies (2 to 5 days old) were individually housed in glass tubes (length, 65 mm; inside diameter, 3 mm) containing standard fly food at one end and a cotton plug on the other end. Sucrose-agar (1% agar supplemented with 5% sucrose) food was used for the GeneSwitch system assay, instead of standard food. The glass tubes were placed in the Drosophila activity monitor (DAM) (Trikinetics, MA, USA) and the locomotor activity of each fly was recorded as the number of infrared beam crossings in 1-min bin. Sleep was defined as periods of inactivity lasting 5 min or longer. Sleep assay were performed for 3 d under LD cycle condition and then constant darkness (DD) conditions. For LD, zeitgeber time (ZT) was used, and for DD, circadian time (CT), with CT 0 as 12 h after lights-off of the last LD conditions, was used to indicate the daily time.

For conditional expression analysis, we used the GeneSwitch system⁴¹ where expression is induced by a steroid hormone antagonist RU486. Flies are monitored for 3 days in tubes without drug in DD and then transferred to new tubes either with vehicle (0.5% DMSO) alone or with 0.5 mM RU486 and then further monitored under DD condition. The expression of endogenous or transgenic Sik3 genes were confirmed by RT-PCR using RNA from fly heads.

Nematode strains and quiescence assay

The wildtype strain N2 and the mutant strain PY1479 kin-29(oy38) X were obtained from the Caenorhabditis Genetics Center (CGC)⁴². All worms were maintained at 20°C on nematode growth medium (NGM) agar plates seeded with E. coli HB101. For construction of P_H20::kin-29, kin-29 cDNA was amplified by RT-PCR and inserted into the plasmid pPD-DEST (a gift from Y. Iino, the University of Tokyo) to generate pDEST-KIN-29. Next, we carried out the LR-recombinase reaction (Gateway System, Life Technologies) between pENTR-P_H20 (a gift from Y. Iino, the University of Tokyo) and pDEST-KIN-29 to generate P_H20::kin-29. P_H20::kin-29 was injected at 30 ng/μl together with the injection marker P_myo-3::mcherry (10 ng/μl) and the empty vector pPD49_26 (60 ng/μl) into the kin-29(oy38) mutant worms.

Quiescence during the L4 to adult lethargus was measured using the microfluidic-chamber based assay⁴³. Briefly, polydimethylsiloxane (PDMS)-made microfluidic chambers containing liquid NGM and the E. coli HB101 were loaded with early L4 larvae and sealed with a cover glass plus 2% agarose, and set under the microscope. Images were taken every 2 seconds for 12 to 20 hours at 20 ± 0.5°C using the microscope M205FA (Leica) equipped with the camera MC120HD (Leica) (pixel size: 1024 μm × 768 μm) controlled by Leica Application Suite V4.3 or the microscope SZX16 (Olympus) equipped with the camera GR500BCM2 (Shodensha) (pixel size: 1024 μm × 768 μm) controlled by μManager (UCSF). Subtraction between serial images was carried out using Image J, and worms were regarded as quiescent at a specific time point if the difference from the preceding time point was less than 1% of the total body size. The fraction of quiescence was defined as the number of quiescent time points divided by the total number of time points during a period of 10 min.

The onset of lethargus quiescence was defined as the time point after which the fraction of quiescence was higher than 0.05 for at least 20 min, whereas the end point was defined as the time point after which the fraction of quiescence was lower than 0.05 for at least 20 min. Occasionally, brief episodes of quiescence were observed outside of lethargus both in wildtype and mutant worms; these episodes were excluded by setting a threshold of 60 min for the minimum duration of lethargus quiescence.

Statistics

Sample sizes were determined using R software based on averages and standard deviations that were obtained from small scale experiments. No method of randomization was used in any of the experiments. The experimenters were blinded to genotypes and treatment assignment. Statistical analysis was performed using SPSS Statistics 22 (IBM) and R software. All data were tested for Gaussian distribution and variance. Homogeneity of variances was tested with Levene’s test. We used Student’s t-test for pairwise comparisons, one-way ANOVA for multiple comparisons, one-way repeated measure ANOVA for multiple comparisons with multiple data points, and two-way ANOVA for multiple comparisons involving two independent variables.

ANOVA analyses were subjected to Tukey’s post hoc test. When deviation from normality and lack of homogeneity of variances occurred (P < 0.05), Mann-Whitney U test was used for group comparison. P < 0.05 was considered statistically significant.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.