(a) Representative images of shControl (Con) and shNEDD9 (N1, N2) MDA-MB-231 cells stained with anti-Rab4 or anti-Rab5 antibodies, DNA (DAPI); Scale bar is 10μm. (b) Quantification of volume of Rab4 and Rab5 positive vesicles based on 3D reconstruction of confocal images; mean volume (%) to control (Con, assigned as 100%) ±S.E.M; three independent experiments, 100 cells/treatment; one-way ANOVA with Dunnett's post-hoc analysis Rab4: *p<0.0001, =0.0002 for Con/N1 or/N2; Rab5:*p<0.0001, =0.0165 for Con/N1 or /N2. (c) Representative image of WB analysis of shControl (Con) and shNEDD9 (N1, N2) MDA-MB-231 cells without (−) or with (+) re-expression of wild type NEDD9 with anti-NEDD9 (2G9) antibodies; GAPDH used as loading control. (d-e) Quantification of volume of Rab4 (d) and Rab5 (e) positive vesicles in cells as in (c) with and without NEDD9 rescue; mean volume as % to control (Con, assigned as 100%) ±S.E.M; three independent experiments, 100 cells/treatment; one-way ANOVA with Dunnett's post-hoc analysis Rab4: *p<0.0001, for Con/N1 or/N2; ns for Con/N1+NEDD9/or N2+NEDD9; Rab5:*p<0.0001 for Con/N1 or /N2; ns for Con/N1+NEDD9/or N2+NEDD9.