Enhancement of JNK-mediated Bim phosphorylation by drug combination treatment. (A) Phosphorylation of JNK and Bim. HeLa and MDA-MB-231 cells were treated with DOX and gamitrinib alone or in combination for 24 hours as indicated and analyzed by western blotting. (B) JNK silencing compromises the synergistic effect of combination treatment. HeLa cells were treated with JNK siRNA prior to treatment with 2 μM DOX and 5 μM gamitrinib as indicated for 24 hours. Cell viability was analyzed by MTT assay. Data are mean ± SEM of two independent experiments performed in triplicate. *, p < 0.02; **, p = 0.0004. (C) Mitochondrial accumulation of Bim and Bax. After treatment of HeLa cells with 5 μM gamitrinib, 2 μM DOX, or 10 μM SP600125 as indicated, mitochondria were fractionated and analyzed by western blotting (left). Bim phosphorylation in the whole cell extract and the mitochondrial fraction (right). (D) Effect of Bim silencing. After treatment with control or Bim-specific siRNAs, HeLa cells were incubated with 2 μM DOX and 5 μM gamitrinib for 24 hours as indicated and analyzed by MTT assay. Data show mean ± SEM of two independent experiments performed in triplicate. ***, p < 0.0001.