Induction of apoptosis by combination treatment. (A) Cell death analysis. 22Rv1 and MDA-MB-231 cells were treated with 0.25 μM DOX/2.5 μM gamitrinib and 10 μM DOX/5 μM gamitrinib, respectively, as single agents or combination treatment for 24 hours, and analyzed for propidium iodide, Sytox, Annexin V staining, and DEVDase activity by flow cytometry. The percentage of cells in each quadrant is indicated. (B) Caspase activation by the drug combination. 22Rv1 cells were treated with 0.25 μM DOX and 2.5 μM gamitrinib, and analyzed by western blotting. The pan-caspase inhibitor zVAD-fmk was used at a concentration of 10 μM. (C) Effect of caspase inhibitor on the drug combination treatment. 22Rv1 cells were treated with 0.25 μM DOX, 2.5 μM gamitrinib, and 10 μM zVAD-fmk as indicated and analyzed by MTT assay. Percent viability compared with DMSO-treated control is shown. Data are the mean ± SEM of duplicated three independent experiments.