The RAB4–RABENOSYN-5 recycling pathway is necessary for HGF-induced invadosome formation. (A) Serum-starved MCF10.DCIS.com cells, transfected with scrambled siRNA (Ctr), siRNAs against RABENOSYN-5 (siRNA RBNS-5) or RAB4A and -B (siRNA RAB4), or RABAPTIN-5. GFP-RAB4AS22N-MCF10.DCIS.com cells were induced or not induced with doxycycline and serum starved. Cells were plated onto fluorescently conjugated gelatin (green), stimulated with HGF for 3 h, or left in serum free conditions (SF), and stained with phalloidin (red). Bar, 15 µm. (A, left) GFP-RAB4AS22N (GFP-RAB4A) expression was verified by immunoblotting. (B) Gelatin degradation was expressed as a degradation index. Data are the mean ± SEM (error bars; n = 75 cells/experiment in four independent ones). *, P < 0.01. Silencing of RAB4A, RAB4B, and RABENOSYN-5 genes was performed using two independent siRNA oligos, which gave identical results (not depicted), and was verified by QRT-PCR (right). **, P < 0.005. (C) MCF10.DCIS.com cells were cotransfected with scrambled siRNA (Ctr) or siRNAs against RABENOSYN-5 (siRNA RBNS-5) together with siRNA-resistant RABENOSYN-5 wild type (resWT-RBNS-5) or mutants impaired in binding to RAB4 (resRAB4-Δ-RBNS-5) or to RAB5 (resRAB5-Δ-RBNS-5), or to both GTPases (resRAB4/5-Δ-RBNS-5) fused to GFP. Cells were plated onto fluorescently conjugated gelatin (red), stimulated with HGF for 3 h, or left in serum free conditions, and stained with DAPI (blue). Arrows, matrix degradation areas. Bar, 20 µm. (D) Gelatin degradation is expressed as a degradation index, as indicated in B. Data are the mean ± SEM (error bars; n = 65 cells/experiment in three independent ones). *, P < 0.01. Silencing of RABENOSYN-5 was verified by QRT-PCR (right). **, P < 0.005.