Fig. 4.

SNX9 negatively regulates invadopodia formation. (A) 231-CTR or 231-siSNX9 cells were plated on gelatin and stained for cortactin (green) and F-actin (phalloidin, in red) to visualize invadopodia. Insets represent enlargement of yellow boxes. Scale bars: 20 μm, insets 4 μm. (B,C) Quantification of percentage of cells expressing invadopodia (B) or of the relative number of invadopodia (C) in conditions used in A. ∼200 cells were counted for each condition, n=3; *P≤0.05, ****P<0.0001. Results are presented as mean±s.e.m. Statistical significance was evaluated using one-tailed Mann–Whitney test. (D) 231-CTR or 231-siSNX9 cells plated on a filter containing 1-μm-diameter pores. Images represent a z-stack of confocal sections of invadopodia extending through the filter. F-actin is visualized in red and cortactin in green. Two independent siRNAs against SNX9 were used. (E) Western blot analysis of SNX9 expression under conditions used in D showing knockdown of SNX9 expression in MDA-MB-231 cells transfected with SNX9 siRNA #1 and #2. GAPDH was used as loading control.