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. 2015 Sep 28;35(23):3049–3061. doi: 10.1038/onc.2015.361

Figure 4.

Figure 4

INPP4B promotes proliferation and anchorage-independent growth of normal colon epithelial cells and modulates colon cancer xenograft growth. (a) Whole-cell lysates from FHC normal colon epithelial 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 and pThr308-Akt), Akt, phosphorylated SGK3 (pThr320-SGK3), SGK3 and GAPDH (as a loading control). Data are representative of three individual experiments. (b) Representative microphotographs of anchorage-independent growth of FHC cells transduced with the pCDH vector alone or INPP4B cDNA cloned into the pCDH vector (pCDH-INPP4B). Data are representative of three individual experiments. Scale bar, 0.5 mm. (c) FHC cells transduced with the pCDH vector alone or INPP4B cDNA cloned into the pCDH vector (pCDH-INPP4B) 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) FHC cells were transduced with the pCDH vector alone or INPP4B cDNA cloned into the pCDH vector (pCDH-INPP4B). Viable cells were counted in an automated cell counter at days 3, 6 and 9 after transduction. Data are represented as mean±s.e.m. of three individual experiments. *P<0.05, Student's t-test. (e) FHC cells transduced with the pCDH vector alone or INPP4B cDNA cloned into the pCDH vector (pCDH-INPP4B) were transduced with shControl, Akt shRNA (shAkt), SGK3 shRNA (shSGK3) or shAKT plus shSGK3. Forty-eight hours later, whole-cell lysates were subjected to western blotting analysis of Akt, SGK3, INPP4B and GAPDH (as a loading control). Data are representative of three individual experiments. (f) FHC cells transduced with the pCDH vector alone or INPP4B cDNA cloned into the pCDH vector (pCDH-INPP4B) were transduced with shControl, shAkt or shSGK3. Forty-eight hours later, cells were subjected to BrdU incorporation assays. Data are represented as mean±s.e.m. of three individual experiments. *P<0.05, Student's t-test. (g) Representative photographs of xenografts of HCT116 cells transduced with shControl or shINPP4B1 in flanks of nu/nu mice (n=8). Mice were euthanized and tumor harvested at 36 days after cell injection. Scale bar, 5 mm. (h) Comparison of growth curves of xenografts of HCT116 cells transduced with shControl or shINPP4B1. Data are represented as mean±s.e.m. of xenografts in 8 mice. *P<0.05; **P<0.01, Student's t-test. (i) Comparison of weight of harvested xenografts of HCT116 cells transduced with shControl or shINPP4B1. Data are represented as mean±s.e.m. of xenografts in eight mice. **P<0.01, Student's t-test. (j) Whole-cell lysates of crude tissues from representative xenografts of HCT116 cells transduced with shControl or shINPP4B1 were subjected to western blotting analysis of INPP4B, phosphorylated Akt (pSer473-Akt), Akt, phosphorylated SGK3 (pThr320-SGK3), SGK3, p27, p21 and GAPDH (as a loading control). Data shown are representative of three individual western blotting analyses of randomly selected tumor samples. (k) HCT116 cells transduced with shINPP4B1 were transduced with the vector alone, myr-Akt cDNA or myr-SGK3 cDNA. Forty-eight hours later, whole-cell lysates were subjected to western blotting analysis of phosphorylated Akt (pSer473-Akt), Akt, phosphorylated SGK3 (pThr320-SGK3), SGK3 and GAPDH (as a loading control). Data shown are representative of three individual experiments. (l) Representative photographs of xenografts of HCT116 cells transduced with shINPP4B1 with or without co-transduction with the vector alone, myr-Akt cDNA or myr-SGK3 cDNA into the flanks of nu/nu mice (n=8). Mice were euthanized and tumor harvested at 36 days after cell injection. Scale bar, 5 mm. (m) Comparison of volume of xenografts of HCT116 cells transduced with shINPP4B1 with or without co-transduction with the vector alone, myr-Akt cDNA or myr-SGK3 cDNA. Data are represented as mean±s.e.m. of xenografts in eight mice. *P<0.05, Student's t-test. (n) Comparison of weight of harvested xenografts of HCT116 cells transduced with shINPP4B1 with or without co-transduction with the vector alone, myr-Akt cDNA or myr-SGK3 cDNA. Data are represented as mean±s.e.m. of xenografts in eight mice. *P<0.05, Student's t-test.