Figure 2

INPP4B promotes colon cancer cell proliferation. (a) Whole-cell lysates from WiDr and HCT116 colon cancer cells and MCF-7 breast cancer cells stably transduced with the control shRNA (shControl) or two individual INPP4B shRNAs (shINPP4B1 and shINPP4B2) were subjected to western blotting analysis of INPP4B, phosphorylated Akt (pSer473-Akt and pThr308-Akt), Akt and GAPDH (as a loading control). Data are representative of three individual experiments. (b) WiDr and HCT116 cells stably transduced with the control shRNA (shControl) or INPP4B shRNA (shINPP4B1) were treated with epidermal growth factor (100 nM) for the indicated periods. Whole-cell lysates were subjected to western blotting analysis of INPP4B, phosphorylated Akt (pSer473-Akt and pThr308-Akt), Akt, and GAPDH (as a loading control). Data are representative of three individual experiments. (c) WiDr, HCT116 and MCF-7 cells were stably transduced with the control shRNA (shControl) or two individual INPP4B shRNAs (shINPP4B1 and shINPP4B2). Forty-eight hours later, cells were subjected to bromodeoxyuridine (BrdU) incorporation assays. Data are represented as mean±s.e.m. of three individual experiments. *P<0.05, Student's t-test. (d) WiDr, HCT116 and MCF-7 cells stably transduced with the control shRNA (shControl) or two individual INPP4B shRNAs (shINPP4B1 and shINPP4B2) were subjected to clonogenic assays. Data are representative of three individual experiments. Scale bar, 1 cm. (e) WiDr and HCT116 cells stably transduced with shControl or shINPP4B1 were transduced with a shRNA-resistant mutant form of INPP4B (INPP4B-mut). Forty-eight hours later, whole-cell lysates were subjected to western blotting analysis of INPP4B and GAPDH (as a loading control). Data are representative of three individual experiments. (f) WiDr and HCT116 cells stably transduced with shControl or shINPP4B1 were transduced with a shRNA-resistant mutant form of INPP4B (INPP4B-mut). Forty-eight hours later, cells were subjected to BrdU incorporation assay. Data are represented as mean±s.e.m. of three individual experiments. *P<0.05, Student's t-test. (g) Whole-cell lysates from SW620 and HT-29 colon cancer cells and MDA-MB-231 breast cancer cells stably transduced with the pCDH vector alone or INPP4B cDNA cloned into the pCDH vector (pCDH-INPP4B) were subjected to western blotting analysis of INPP4B, phosphorylated Akt (pSer473-Akt), Akt and GAPDH (as a loading control). Data are representative of three individual experiments. (h) SW620, HT-29 and MDA-MB-231 cells stably transduced with the pCDH vector alone or INPP4B cDNA cloned into the pCDH vector (pCDH-INPP4B) were subjected to clonogenic assays. Data are representative of three individual experiments. Scale bar, 1 cm.