Podocalyxin promotes tumorsphere formation. (A)
PODXL gene expression relative to GAPDH in scrambled shRNA (shCTRL) and shRNA targeting PODXL (shPODXL) MDA-MB-231 cells as determined by quantitative PCR (n = 3; *P < 0.05; ***P < 0.001). (B) Podocalyxin expression on shCTRLGFP cells (green fluorescent protein (GFP); solid green) and shPODXLGFP (dashed green) MDA-MB-231 cells (left histogram) and shCTRLRFP (red fluorescent protein (RFP); solid red) and shPODXLRFP (dotted red) MDA-MB-231 cells (right histogram) relative to isotype control (shaded). RFI, Relative fluorescence intensity. (C) Tumorsphere-forming efficiency of shCTRL (solid bar) and shPODXL (open bar) MDA-MB-231 cells cultured for 7 days under anchorage-independent conditions (n = 3; **P < 0.01, ***P < 0.001). (D) Self-renewal capacity of shCTRLRFP (solid bars) and shPODXLRFP (open bars) MDA-MB-231 tumorspheres assessed by serial passaging (P) (n = 3; ***P < 0.001 by one-way analysis of variance). (E) MCF7pIRES control (solid bar) and MCF7Podxl (open bar) cells were cultured under anchorage-independent conditions for 7 days to assess tumorsphere-forming efficiency (n = 3; **P < 0.01). Figures are representative of three experiments with similar results. Error bars indicate standard error of the mean.