Effects of single agent and combination therapy on apoptosis. To test the cytotoxic effects of the different treatments, melanoma cells were treated for 72 hours with 5 μM vemurafenib, 10 mM metformin, or the combination. Cells were stained with Annexin V and PI and were analyzed by flow cytometry. a) Annexin/PI staining of M263 as a representative example. Upper left corner: DMSO, upper right corner: vemurafenib, lower left corner: metformin, lower right corner: Combination of vemurafenib and metformin. b) Early and late apoptotic cells after 72-hour treatment with the indicated agents. Percentages are shown in relation to the control, DMSO treated cells.