Figure 6

Effect of γ-actin knockdown on cell adhesion, migration and motility. (A) Histogram showing the relative adhesion of endothelial cells following treatment with control (white) and γ-actin siRNA (black) for 72 h. Fluorescently labeled HMEC-1 cells were allowed to adhere to various substrates for 1 h. Columns, means of at least three individual experiments; bars, SE. (B) Histogram showing the relative migration of endothelial cells towards FCS, VEGF, FGF_β and ECGF following treatment with control (white) and γ-actin siRNA (black) for 72 h. Fluorescently labeled HMEC-1 cells were allowed to migrate through 8 μm pore PET membrane and towards a chemo-attractant for 6 h. Columns, means of at least four individual experiments; bars, SE. Statistics were calculated by comparing the fluorescence measured at 492/517 (Abs/Em) with control and γ-actin siRNA-treated cells; *, p < 0.05; **, p < 0.001. (C) Representative trajectories of 5 individual HMEC-1 cells, recorded by time-lapse videomicroscopy over 6 h, following treatment with control (left) and γ-actin siRNA (right) for 72 h. Scale, −130 μm to +130 μm for both x and y axes. (D) Representative photographs of control (top) and γ-actin siRNA-treated (bottom) BMH29L cells in wound healing experiments, taken 12 h after start of experiment. Broken lines show the position of the initial cell-free gap (at time 0) and solid lines highlight the position of the migration edge after 12 h. Inset, % of wound closure. (E) Graph showing the percentage of wound recovery as a function of time for control (black, solid line) and γ-actin siRNA-treated (red, broken line) BMH29L cells. Points, means of at least four individual experiments; bars, SE. Statistics were calculated by comparing control and γ-actin siRNA-treated cells at specific time points; *, p < 0.05; **, p < 0.01; ***, p < 0.001.