Fig. 4

Induction of apoptosis through cytochrome c release with combined metformin and OSMI-1 treatment. A Flow cytometry analysis of cytochrome c release showing the fluorescence intensity of the FITC-A channel in HCT116 cells. Cells were treated with metformin (25 mM) alone or in combination with OSMI-1 (20 μM) for 48 h and the amount of cytochrome c was analyzed by flow cytometry. B HCT116 cells were treated with metformin (25 mM), OSMI-1 (20 μM), or a combination thereof for 48 h. After staining with Annexin V-FITC/PI, the level of apoptosis was determined by flow cytometry. The proportion of early and late apoptotic cells was compared. C HCT116 cells were transfected with OGT siRNA and treated with metformin (25 mM) alone or a combination thereof for 48 h. The level of cleaved caspase-3 was determined by western blot analysis. β-Actin was used as a loading control in all western blot analyses. D HCT116 cells were transfected with CHOP siRNA and incubated with SP600125 alone or a combination thereof for 1 h and then treated with a combination of metformin (25 mM) and OSMI-1 (20 μM) for 48 h. After staining with annexin V-FITC/PI, the level of apoptosis was determined by flow cytometry. The proportion of early and late apoptotic cells was determined. B, D Experiments were performed in triplicate and data represents the mean ± SEM. Significance was determined by control and combination group Student’s t-test. ***p < 0.001