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. 2015 Oct 19;1:15044. doi: 10.1038/cddiscovery.2015.44

Figure 6.

Figure 6

The H1047R mutation in the p110α kinase domain of PI3K affected Bcl-2 expression level. (a) Endogenous level of Bcl-2 in HCT116 WT and MUT cells. Endogenous level of Bcl-2 in HCT116 WT and MUT cells was measured by immunoblotting analysis (top). The graph shows the quantification of Bcl-2 bands normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for three individual experiments (bottom). The Bcl-2 level in HCT116 WT cells was at least three times higher than in HCT116 MUT cells. (b) Effects of overexpressing WT-p110α and H1047R-p110α on Bcl-2 levels. HCT116 WT cells, which were transfected with the Tet-On 3G-inducible plasmids pTRE3G-BI-mCherry/p110αWT or pTRE3G-BI-mCherry/p110αH1047R, were cultured in the medium containing indicated concentration of doxycycline (DOX; left). Their Bcl-2 levels were measured by immunoblotting analysis (left). The graph shows the quantifications of Bcl-2 bands of two different clones from the individual cell lines (right). Overexpression of WT-p110α resulted in a DOX dose-dependent increase in Bcl-2 levels, however, Bcl-2 levels were not verified by overexpression of H1047R-p110α. (c) Effect of p110α inhibition on Bcl-2 levels. HCT116 WT and MUT cells were serum starved overnight, subsequently cultured in the presence of A66 at the indicated concentrations for 3 h, and then followed by 100 ng/ml EGF stimulation for 20 min. Immunoblotting analysis was used to measure Bcl-2 levels in HCT116 WT (left) and MUT (right) cells. The graph shows the quantifications of expression Bcl-2 levels in HCT116 WT and MUT cells on treatment of A66 (bottom panel). Data are the average of two independent experiments. Bcl-2 levels in HCT116 MUT cells was A66 dose-dependently increased, however, an A66 dose-dependent decrease was observed in HCT WT cells.