Rab31 promotes MCF-10A mammosphere formation by a MUC1-C-dependent mechanism.

A. The indicated MCF-10A cells were seeded in soft agar. After culturing for 3 weeks, photomicrographs were obtained (left) and the numbers of colonies (mean±SD of three replicates) were determined by counting clusters of >20 cells. B. The indicated MCF-10A cells were seeded under conditions for the growth of mammospheres. After culturing for 3 weeks, photomicrographs were obtained (left) and the numbers of mammospheres (mean±SD of three replicates) were determined by counting spheres of >20 cells (right). C. Lysates from MCF-10A/Rab31 cells grown as an adherent monolayer and as mammospheres were immunoblotted with the indicated antibodies (left). MCF-10A/Rab31 cells were transfected with control and MUC1 siRNAs. Lysates were immunoblotted with the indicated antibodies (right). D. MCF-10A/Rab31 cells transfected with the indicated siRNAs were cultured for the growth of mammospheres. After 3 weeks, photomicrographs were obtained (left) and the numbers of mammospheres (mean±SD of three replicates) were determined by counting spheres of >20 cells (right). E. MCF-10A/Rab31 cells were cultured for the growth of mammospheres in the absence (Control) and presence of 5 µM GO-203 or CP-2. After 3 weeks, photomicrographs were obtained (left) and the numbers of mammospheres (mean±SD of three replicates) were determined by counting spheres of >20 cells (right).