Cdc42 drives linear invadosome formation via DDR1. (A) Western blot analysis of MDA-MB-231 cells transfected with siRNA control (siCT) or two independent siRNAs targeting Rac1, Cdc42, or RhoA. GAPDH is used as a loading control. Three independent experiments were realized and quantified to demonstrate a specific effect on targeted RhoGTPase expression represented on the bar graph on the right. (B and C) MDA-MB-231 cells transfected as in A were cultured on collagen I for 4 h, fixed, and processed for immunofluorescence staining. (B) Representative confocal images of MDA-MB-231 cells transfected as in A. Tks5 (green) and F-actin (red) are shown. Panels on the right show enlarged views of the boxed regions. Bars: (left) 10 µm; (enlarged panels on the right) 2.5 µm. (C) The percentage of siRNA-transfected MDA-MB-231 cells able to form linear invadosomes was quantified. Error bars represent the SEM (n > 1,000, three independent experiments; ***, P < 0.001 as compared with the control siRNA condition [siCT]).