Figure 4: MiR-708 inhibits FA formation, cell spreading and cell adhesion.

(a) Confocal microscopy of Paxillin (Green), β-actin (Red) and DAPI (Blue) nuclear staining, after adhering of cells to FN. SKOV-I6iv cells with different transfections were equally plated on FN-coated coverslips for 90 min. The FAs were detected. Scale bars: 10 μm. (b–e) The images were analysed to determine the number of FAs per cell (b), the area of the cell staining for FAs by paxillin staining (c) and the cell-spreading area by β-actin staining (d). To determine the proportion of each cell consisting of FAs, the area of FAs was divided by the total spreading area of the cell (e). Data in b–e represent means±s.e.m. (n=30). (f) Left: western blot analysis showing p-Paxillin, p-FAK, Paxillin and FAK expressions in SKOV-I6iv cells with different transfections. Cells were incubated on FN-coated plates for 60 min before being subjected to western blot analysis. Right: quantitative analysis of protein levels, and normalized with total FAK or paxillin levels. Histograms represent normalized means±s.e.m. (n=3). (g) Adhesion assays of SKOV-I6iv cells with GFP plus different transfections. Percentage of adhered cells was counted. Data represent means±s.d. (n=3). Student’s t-test (two-tailed) was used for statistical analysis (*P<0.05; **P<0.01; ***P<0.001).