TKS5 is required for invadopodia formation and function. (A) MDA-MB-231 cells were cultured on a fibrillary layer of type I collagen (gray) for 60 min and stained for TKS5 (red), F-actin (green), and DAPI (blue). (B) MDA-MB-231 cells expressing TKS5^GFP (green) were cultured on a collagen layer (gray) for 60 min. Cleaved collagen was stained with the Col1-^3/4C antibody (red). Cell nuclei were stained with DAPI (blue). Scale bar, 10 µm; zoom-in of boxed region, 5 µm. (C) Cells depleted for TKS5 were analyzed as in A. (D) Quantification of TKS5-positive invadopodia in siRNA-treated MDA-MB-231 cells. The y axis is the ratio of the TKS5 area to the total cell area normalized to the mean value of control siNT-treated cells (as percentage ± SEM). siNT, n = 73 cells; siTKS5, n = 66 cells from three independent experiments. Data were analyzed using t test. (E) Pericellular collagenolysis by the indicated MDA-MB-231 cell populations measured as mean intensity of Col1-^3/4C signal per cell ± SEM. Values for control cells were set to 100%. siNT, n = 61 cells; siMT1-MMP, n = 63 cells; and siTKS5, n = 42 cells from three independent experiments. Kruskal–Wallis test. ****, P < 0.0001.