Linear invadosome formation and activity is independent of Src activity. (A and B) MDA-MB-231 cells were seeded on gelatin-FITC (top) or on a mixed matrix (collagen I/gelatin-FITC; bottom) and treated with 5 µM PP2 (Src inhibitor) or DMSO (vehicle). Gelatin (gray), Tks5 (green), and F-actin (red) are shown. (C) Quantification of the degradation capacity of MDA-MB-231 cells seeded on a mixed gelatin/collagen I matrix treated or not treated with PP2. The left graph represents the gelatin area degraded per cell after 24 h. The right graph represents the amount of collagen degraded after 4 h (n = 30 fields). Data are shown as mean ± SEM of three independent experiments. (D) MDA-MB-231 cells transfected with control (siCT) or two independent DDR1 siRNAs (DDR1 #1 and #2) were seeded on gelatin or collagen I and treated with PP2. Protein extracts were then analyzed by immunoblotting to determine phospho-Src, total Src, and DDR1 protein expression (representative of three experiments). (E and F) Western blots performed on SYF and SYF c-Src protein extracts representing, respectively, protein expression of Src and DDR1. GAPDH was used as a loading control. (G and H) Confocal images of control cells (SYF c-Src) and SYF fibroblasts cultured on gelatin (G) or on a mixed matrix (gelatin/collagen I; H) for 24 h and processed for immunofluorescence staining (F-actin, red; Tks5, green; DAPI, blue). Insets on the bottom show gelatin-degraded pictures. Bars: (A, G, and H) 10 µm; (A, insets) 7 µm; (H, top insets) 2 µm; (G and H, bottom insets) 10 µm.