HGF-induced fast recycling of MT1-MMP depends onto RAB5, RAB4, and RABENOSYN-5. (A) Cherry-MT1-MMP–expressing HeLa cells were transfected with scrambled siRNA or siRNAs against RAB5A,B,C (siRNA-RAB5) or RAB4A and -B (siRNA RAB4 A/B), RABENOSYN-5 (siRNA RBNS-5), or integrin β3 (siRNA β3). Serum-starved cells were incubated with anti–MT1-MMP antibody at 16°C for 2 h. After a mild acid wash to remove surface antibody, cells were switched to 37°C and stimulated with HGF (100 ng/ml), or left in serum free (SF) conditions. At the indicated time points, cells were fixed, and stained in the absence of permeabilization with FITC-conjugated secondary antibody (green). Cherry-MT1-MMP (red) was detected by epifluorescence. The relative cell surface levels of MT1-MMP were quantified using ImageJ software on nonsaturated images, and expressed as relative cell surface MT1-MMP levels with respect to HGF-stimulated control cells after 15 min (set at 100%). Data are the mean ± SEM (error bars; n = 25 cells repeated in three independent experiments). (B) Steady-state cell surface levels of MT1-MMP. Cherry-MT1-MMP-HeLa cells were transfected with scrambled siRNA or siRNAs against RAB5A,B,C (siRNA-RAB5) or RAB4A and -B (siRNA RAB4 A/B), RABENOSYN-5 (siRNA RBNS-5), or integrin β3 (siRNA β3). Cells were incubated with anti–MT1-MMP antibody at 4°C for 2 h, washed, fixed, and stained in the absence of permeabilization with FITC-conjugated secondary antibody (green). (C) Silencing of RAB5 impairs MT1-MMP internalization. Cherry-MT1-MMP-HeLa cells were transfected with scrambled (Ctr) or anti-RAB5A,B,C siRNAs. Serum-starved cells were incubated with anti–MT1-MMP antibody at 16°C for 2 h. Cells were washed, fixed, permeabilized with 0.1% Triton X-100, and stained with FITC-conjugated secondary antibody (green). Bottom right, efficacy of gene silencing by QRT-PCR. Data are the mean ± SEM (error bars). **, P < 0.005. Bars, 10 µm.