Figure 5

INPP4B downregulates PTEN in colon cancer cells. (a) The relative abundance of PI(3,4,5)P₃ in WiDr and HCT116 cells transduced with the control shRNA (shControl), INPP4B shRNA (shINPP4B1), PTEN shRNA (shPTEN) or shINPP4B1 plus shPTEN was measured by using ELISA in lipid extractions of whole cells. Data are represented as mean±s.e.m. of three individual experiments. **P<0.01, Student's t-test. (b) The relative abundance of PI(3,4)P₂ in WiDr and HCT116 cells transduced with shControl, shINPP4B1, shPTEN or shINPP4B1 plus shPTEN was measured by using ELISA in lipid extractions of whole cells. Data are represented as mean±s.e.m. of three individual experiments. *P<0.05, Student's t-test. (c) The relative abundance of PI(3)P in WiDr and HCT116 cells transduced with shControl, shINPP4B1, shPTEN or shINPP4B1 plus shPTEN was measured by using ELISA in lipid extractions of whole cells. Data are represented as mean±s.e.m. of three individual experiments. *P<0.05, Student's t-test. (d) Whole-cell lysates from WiDr and HCT116 cells stably transduced with shControl or two INPP4B shRNAs (shINPP4B1 and shINPP4B2) were subjected to western blotting analysis of PTEN, PIB5PA, SHIP2 and GAPDH (as a loading control). Data are representative of three individual experiments. (e) Whole-cell lysates from WiDr and HCT116 cells stably transduced with shControl or shINPP4B1 were subjected to immunoprecipitation with a rabbit antibody against PTEN or control rabbit IgG. The precipitates were subjected to western blotting analysis of PTEN. Data are representative of three individual experiments. (f) Whole-cell lysates from WiDr and HCT116 cells stably transduced with shControl or shINPP4B1 were subjected to immunoprecipitation with a rabbit antibody against PTEN. The precipitated PTEN was subjected to analysis of PTEN lipid phosphatase activity using a PTEN activity ELISA kit. The relative PTEN lipid phosphatase activity in cells transduced with shControl was arbitrarily designated as 1. Data are represented as mean±s.e.m. of three individual experiments. *P<0.05, Student's t-test. (g) Whole-cell lysates from WiDr and HCT116 cells transduced with shControl, shINPP4B1, shPTEN or shINPP4B1 plus shPTEN were subjected to western blotting analysis of INPP4B, PTEN, hosphorylated Akt (pSer473-Akt), Akt, phosphorylated SGK3 (pThr320-SGK3), SGK3 and GAPDH (as a loading control). Data are representative of three individual experiments. (h) WiDr and HCT116 cells were transduced with shControl, shINPP4B1, shPTEN or shINPP4B1 plus shPTEN. Forty-eight hours later, cells were then 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.