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. 2015 Nov 9;6(37):39891–39907. doi: 10.18632/oncotarget.5359

Figure 2. INPP4B promotes proliferation of melanoma cells independently of Akt.

Figure 2

A. Whole cell lysates from Mel-RM and ME4405 melanoma cells and MCF-7 breast cancer cells stably transduced with the control shRNA (shControl) or two INPP4B shRNAs (shINPP4B1 and shINPP4B2) were subjected to Western blot analysis of INPP4B, phosphorylated Akt (pSer473-Akt), Akt and GAPDH (as a loading control). The data shown are representative of three individual experiments. B. Mel-RM, ME4405 and MCF-7 cells were stably transduced with the control shRNA (shControl) or two INPP4B shRNAs (shINPP4B1 and shINPP4B2). Forty-eight hours later, cells were subjected to BrdU incorporation assays. The data shown are mean ± SEM of three individual experiments. *P < 0.05, Student's t-test. C. Mel-RM, ME4405 and MCF-7 cells stably transduced with the control shRNA (shControl) or two INPP4B shRNAs (shINPP4B1 and shINPP4B2) were subjected to clonogenic assays. The data shown are representative of three individual experiments. Scale bar, 1cm. D. Mel-RM and ME4405 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 blot analysis. The data shown are representative of three individual experiments. E. Mel-RM and ME4405 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 the BrdU incorporation assay. The data shown are mean ± SEM of three individual experiments. *P < 0.05, Student's t-test. F. Whole cell lysates from MM200 and Mel-RM melanoma cells and MDA-MB-231 breast cancer cells stably transduced with the vector alone or INPP4B cDNA cloned in the pCDH vector (pCDH-INPP4B) were subjected to Western blot analysis of INPP4B, phosphorylated Akt (pSer473-Akt), Akt and GAPDH (as a loading control). The data shown are representative of three individual Western blot analyses. G. MM200, Mel-RM and MDA-MB-231 cells stably transduced with the vector alone or INPP4B cDNA cloned in the pCDH vector (pCDH-INPP4B) were subjected to BrdU incorporation assays. The data shown are mean ± SEM of three individual experiments. *P < 0.05, Student's t-test. H. MM200, Mel-RM and MDA-MB-231 cells stably transduced with the vector alone or INPP4B cDNA cloned in the pCDH vector (pCDH-INPP4B) were subjected clonogenic assays. The data shown are representative of three individual experiments. Scale bar, 1cm. I. Mel-RM and ME4405 cells stably transduced with shControl or shINPP4B1 were transduced with the vector alone or myr-Akt cDNA. Forty-eight hours later, whole cell lysates were subjected to Western blot analysis of phosphorylated Akt (pSer473-Akt), Akt and GAPDH (as a loading control). The data shown are representative of three individual Western blot analyses. J. Mel-RM and ME4405 cells stably transduced with shControl or shINPP4B1 were transduced with the vector alone or myr-Akt cDNA. Forty-eight hours later, cells were subjected to BrdU incorporation assays. The data are mean ± SEM of three individual experiments.