Fig. 1

Suppression of RAB4A abolishes tumor formation by inhibiting cancer cell self-renewal/stemness. A, B Orthotopic mammary fat pad tumor formation study. A Shown are the growth rates of tumors derived from MDA-MB-231 breast cancer cells expressing control shRNA (N = 10), or shRNA targeting RAB4A KD#1 or RAB4A KD#2 (5 mice in each group, totaling N = 10). Since no tumors formed in the KD groups, all ten tumor growth time points fall on the X-axis. B Representative in situ tumor images from the study described in (A). The cells were implanted in the mammary fat pad in NOD-SCID mice. The in vivo tumor formation study comparing control and RAB4A KD cells was biologically repeated with similar outcome. C Cell proliferation under adherent culture conditions of MDA-MB-231 cells expressing control shRNA or two different RAB4A shRNAs as indicated. Proliferation was monitored and recorded continuously for 120 h using IncuCyte live-cell imaging. D Serial replating sphere formation assay on the same cell groups as in C for self-renewal/stemness analysis. Top: representative microscopic images of the third replating (Gen-3) spheres; bottom: quantification of relative sphere numbers from second replating (Gen-2) and third replating (Gen-3) assays. The data was analyzed using OpenCFU and Prism software. *p < 0.05