Integrin trafficking promotes invasive migration through the suppression of Rac activity and activation of RhoA. (A) A2780 cells were subjected to control, Rac1, or RhoA SMARTpool RNAi and seeded onto CDMs after 24–36 h. Cells were stimulated with cRGDfV as indicated, and images were captured as in Fig. 2 A. Representative images are shown. Bar, 50 µm. (B–D) A2780 cells were cotransfected with GFP-Rac1 or GFP-RhoA (as indicated) alongside control, Rac1 #1, or RhoA #1 RNAi oligos and seeded onto CDM as in A, and images were captured as in Fig. 2 A. (B) Pseudopod length (>30 cells/condition) was measured as in Fig. 2 A. (C and D) Speed and persistence (≥26 cells/condition) of migration was analyzed using ImageJ. (E) A2780 cells were subjected to control, Rac1, or RhoA SMARTpool RNAi and seeded into inverted invasion assays in the presence of FN and cRGDfV. (F and G) MDA-MB-231 (F) or H1299-vector (VEC)/273H cells were subjected to control, Rac1, or RhoA SMARTpool RNAi and seeded into inverted invasion assays in the presence of FN. Data represent means ± SEM from at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.