Fig. 5.

N-WASP is critical for hypoxia induced invadopodia formation. (A) Western blot analysis of N-WASP and HiF1α expression in MDA-MB-231 cells grown in presence of DMOG for 6 h (B) Densitometric analysis of N-WASP/GAPDH protein ratio in MDA-MB-231 cells treated with DMOG (C) Western blot analysis of N-WASP expression in control cells (MDA-MB-231^Vect), N-WASP knockdown cells reconstituted with N-WASP^R (MDA-MB-231^KD+N-WASP^R) and N-WASP knockdown cells with Vector (MDA-MB-231^KD+Vect) (D) Densitometric analysis of N-WASP/GAPDH protein ratio of western blot in panel C. (E) Cells (MDA-MB-231^Vect, MDA-MB-231^KD+Vect and MDA-MB-231^KD+N-WASP^R) were seeded on fluorescent gelatin coated coverslips and treated with 1 mM DMOG for 6 h, fixed and immunostained. Degraded areas of fluorescent gelatin along with staining for F-actin (red) indicate presence of invadopodia. Images were taken using 40X oil objective lens. Presence of actin dots (red) with underlying degraded gelatin (black areas) was used to identify degradation areas (red dots on gelatin degraded areas). Arrows indicate areas of gelatin degradation (F) Percentage of cells (Panel E) with invadopodia was counted for DMSO and DMOG treated cells and 30 cells were analyzed for each triplicate sample (N=3) *p<0.05; **p<0.01; ***p<0.001.