Invadopodia reformation is impaired in Protrudin-depleted cells. (A) MDA-MB-231 and MDA-MB-231-GFP-Protrudin-sires#1 cells 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 serum free medium (SFM) as a negative control. Cells were stained with antibodies against TKS5 and analyzed by high-content microscopy. Note that TKS5-positive invadopodia only reform in the serum and HGF-treated cells and that cells expressing siRNA-resistant GFP-Protrudin reform invadopodia comparable to control cells. Graphs represent quantifications of different features of invadopodia reformation. Each plotted point represents the mean value of one experiment. Values represent mean ± SD. *, P < 0.05, one-way ANOVA, Tukey’s post hoc test. n = 3 independent experiments. In total, >2,800 cells were analyzed per condition. (B) Graphs represent quantifications of different features of invadopodia reformation from Fig. 2 B. Each plotted point symbolizes one image representing the average value of typically 10–15 cells. Values represent mean ± SD. *, P < 0.05; ***, P < 0.001, one-way ANOVA, Tukey’s post hoc test. n = 25 images per condition from three independent experiments. (C) Real-time PCR for verification of SYT7 knockdown from three independent experiments in MDA-MB-231 cells. **, P < 0.01, one-sample t test.