RAB5A is essential for HGF-induced invadosome formation. (A and B) Doxycycline-induced control and RAB5AS34N-MDA-MB-231 (A) or RAB5AS34N-MCF10.DCIS.com (B) cells plated onto fluorescently conjugated gelatin were stimulated with HGF (100 ng/ml) for 3 h. (B, left) Images of cells stained with phalloidin (left), fluorescently conjugated gelatin (middle), and merged channels (right). (B, right) Quantification of gelatin degradation was expressed as a degradation index (calculated as described in Materials and methods). Data are the mean ± SEM (error bars; n = 70 cells/experiment in five independent ones). **, P < 0.005. (C, left) TIRF microscopy of RAB5A-HeLa cells. F-actin and RAB5A were detected with phalloidin and anti–RAB5 Ab (RAB5), respectively. (C, right) xz sections of control and RAB5A-HeLa cells plated onto fluorescently conjugated gelatin (red) and stained with phalloidin (green). (D) Control and RAB5A-HeLa cells were plated onto fluorescently conjugated gelatin (middle) overnight under serum-starved conditions and stimulated with suboptimal doses of HGF (1 ng/ml). F-actin and RAB5A were detected with phalloidin and anti-RAB5A Ab (RAB5A), respectively. Insets show magnifications of the boxed regions. Arrows indicate invadosomes. (D, right) Gelatin degradation was expressed as a degradation index (see Materials and methods). Data are the mean ± SEM (n = 50 cells/experiment in four independent ones). **, P < 0.005. Bars: (A–C) 20 µm; (D) 10 µm.