Fig. 3

RAB4A regulates RAC1 activation, which is essential for EMT and cell invasion. A qPCR analysis of the expression of ZEB1 and CDH1 in response to RAC1 knockdown. “*” represents p < 0.05 relative to the expression in control cells, which is set as baseline. B Invasion assay assessing the effect of RAC1 knockdown. C Pulldown of GTP-bound RAC1 to study the activation status of RAC1 in response to RAB4A knockdown. Lane 1, 4 and 7–lysate incubated with GTPγS; lane 2, 5 and 8–lysate incubated with GDP; lane 3, 6 and 9–lysate only to assess endogenous level of GTP-bound RAC1; lane 10–recombinant RAC1 as immunoblot control. Total lysate input for three cell lysates: lane 11–Control^KD, lane 12-RAB4A^KD#1, and lane 13-RAB4A^KD#2. β-Tubulin is the loading control. D Invasion assay to assess the rescue effect of ectopic RAC1 expression in RAB4A knockdown MDA-MB-231 cells. E qPCR analysis of RAB4A, ZEB1 and CDH1 expression in the cells in (D). Gene expression levels in control shRNA expressing cells set the baseline. F Immunofluorescence analysis of CDH1 (E-cadherin) expression in MDA-MB-231 cells with RAB4A KD alone with and without RAC1 expression. DAPI was use to stain the nucleus. Data in the bar graphs of A and E are presented as mean ± SEM (n ≥ 5). “*” and “ns” represent p < 0.05 and not significant, respectively, in the comparison to baseline of control cells defined by the x-axis. For invasion study and fluorescence images (B, D and E): scale bar = 100 µM