Figure 4.
Ectopic ZNF281 induces EMT, migration and invasion in DLD-1 cells. DLD-1 cells harbouring a pRTR-ZNF281-VSV vector treated with DOX or left untreated were analysed. (A) Representative phase-contrast (P/C) pictures of the cells treated with DOX or left untreated for 96 h. × 200 magnification. (B) Confocal laser-scanning microscopy of E-cadherin, Vimentin and F-actin proteins detected by indirect immunofluorescence 96 h after addition of DOX. Nuclear DNA was visualized by DAPI staining. × 200 magnification. (C) Cells were treated with DOX or left untreated for 48 h before the scratch was applied. Upper panel: representative pictures of the wound areas at the indicated time points after scratching. × 100 magnification. Lower panel: results represent the average (%) of wound closure determined by the final width of the scratch in three independent wells. Error bars represent±s.d. (n=3). Boyden-chamber assays of cellular migration (D) or invasion (E). Cells were cultivated in the presence or absence of DOX for 72 h with serum starvation for the last 48 h. To analyse invasion, membranes were coated with Matrigel. After 48 h, cells were fixed and stained with DAPI. The average number of cells per well was counted in three different inserts. Relative invasion or migration is expressed as the value of treated cells to control cells with control set as one. (F) Cells were subjected to a soft-agar assay and treated with DOX or left untreated. Two weeks after seeding the resulting colonies were stained with crystal violet. Results represent the mean number of colonies in soft agar per well±s.d. (n=3). (C–F) A Student’s t-test was used. **P<0.01 and ***P<0.001. In (A, B), scale bars represent 25 μm.