TKS5 is required for collagenolysis and for 3D collagen invasion by breast cancer cells. (A) MDA-MB-231 cells were cultured on a fibrillary layer of type I collagen (gray) for 60 min and stained for cortactin (red), TKS5 (green), and DAPI (blue). Scale bar, 10 µm; zoom-in of boxed region, 5 µm. (B) Upper panel: Fluorescence (Fluo) intensity profiles for cortactin and TKS5 were recorded along the invadopodium (cyan dashed line in the insets) and normalized to the maximum fluorescence intensity set to 100. Lower panel: Correlation of cortactin (x axis) and TKS5 (y axis) pixel fluorescence intensity along 28 invadopodial structures. inv, number of invadopodia analyzed; pix, total number of pixels analyzed for both markers. (C) Representative images of pericellular collagenolysis detected with Col1-^3/4C antibody (black signal in the inverted images). Nuclei were stained with DAPI (red). Scale bar, 50 µm. (D) 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%. Mann–Whitney U tests. (E) siRNA-treated cells were allowed to invade type I collagen plugs in an inverted invasion assay. After 48 h of invasion, nuclei were stained with DAPI (green), and serial optical sections (10-µm interval) were acquired. Scale bar, 100 µm. (F) Relative invasion of cells penetrating 3D collagen to depths ≥30 µm (see E). Data represent mean ± SEM normalized to invasion of mock-treated cells from three independent experiments. Kruskal–Wallis test. ****, P < 0.0001.