Protrudin-depleted cells fail to regrow invadopodia. (A) MDA-MB-231 and MDA-MB-231-GFP-Protrudin-sires#1 cells grown on coverslips were transfected with siRNA targeting Protrudin (oligo #1) or control siRNA. 4 d after transfection, cells were serum starved for 4 h and treated with Src inhibitor (10 µM PP2) for the last 30 min to remove invadopodia. Cells were stimulated for 1 h with serum containing medium supplemented with HGF (50 ng/ml) to allow reformation of invadopodia or SFM as a negative control. Cells were stained with antibodies against TKS5 and cortactin and analyzed by confocal microscopy. Micrographs show reformation of TKS5-positive invadopodia in serum- and HGF-treated cells. Note that cells expressing siRNA-resistant GFP-Protrudin reform invadopodia as in control cells. Graphs represent quantifications of different features of invadopodia reformation. Each plotted point symbolizes one image representing the average value of typically 15–20 cells. Values represent mean ± SD. *, P < 0.05; ***, P < 0.001, one-way ANOVA, Tukey’s post hoc test. n = 9 (serum starvation) or n = 15 images per condition from three independent experiments. (B) MDA-MB-231 and MDA-MB-231-GFP-Protrudin-sires#1 cells were siRNA transfected with siRNA targeting Protrudin (oligo #1) or control siRNA. 4 d after transfection, the cells were seeded on unconjugated-gelatin coated coverslips in SFM for 2 h to allow attachment before treating with Src inhibitor (10 µM PP2) for 30 min. Serum containing medium with HGF (50 ng/ml) was added and cells were allowed to reform invadopodia for 4 h, immunostaining and confocal imaging as in A. Note that cells expressing siRNA-resistant GFP-Protrudin reform invadopodia as in control cells (GFP not shown). For quantifications of micrographs, see Fig. S2 B. n.s, not statistically significant.