Fig. 4

RAC1 is the major downstream effector of RAB4A regulation of cancer stemness. A, B Serial replating sphere formation assay on MDA-MB-231 cells with RAB4A knockdown alone and in combination with RAC1 overexpression. A Representative microscopic images of the third-generation (Gen-3) replating spheres. B Quantification of Gen-3 sphere numbers analyzed using OpenCFU and Prism software. *p < 0.05. C, D Fluorescent microscopic analysis of widely used stemness markers of breast cancer cells (CD44, ALDH1A3) on the same cell groups as in (A). Representative images are shown for CD44 staining C and ALDH1A3 staining D. DAPI was used to stain the nucleus. Scale bar = 100 µM