Figure 5.

Effect of miR-183 on the number of focal adhesions. (A) Effect of ITGB1 blockade on cell adhesion. Untransfected cells were incubated with an antibody against ITGB1 (ab24693 Abcam) or TWIST (ab50887 Abcam), seeded on polystyrene 96-well plates and allowed to adhere for 0.5 or 1 h (depending on the cell line). Non-adherent cells were removed, and the remaining cells were quantified using CellTiter-Glo (Promega). The results of at least three independent replicates were normalized to the control and are shown as means ± SD. An unpaired t-test was applied to assess the statistical significance of the difference between conditions. (B) Immunocytochemistry of vinculin of prostate cells overexpressing miR-183. Seventy-two hours after transfection of 20 nM of miR-183, the cells were fixed, permeabilized and immunostained for vinculin using an Alexa Fluor 488-conjugated secondary antibody. Nuclei and filamentous actin were counterstained with DAPI and phalloidin-Alexa Fluor 568, respectively. The images were collected on a confocal microscope using a 63X oil immersion objective. Scale bars show 25 μm. Representative images are shown. (C) Effect of miR-183 on focal adhesion density. Quantification of focal adhesions per cell in vinculin immunocytochemical images of the DU145 and RWPE-1 cell lines. Images were processed using ImageJ. A Mann–Whitney test was applied to test the statistical significance of the difference between conditions. p-value ** <0.01, *** <0.001, **** <0.0001, ns = not significant.