Figure 3

Effect of drug combination on the expression of CHOP and Bim. (A) CHOP induction and JNK phosphorylation. 22Rv1 cells were treated with DOX and gamitrinib alone or in combination as indicated and the whole cell lysate was analyzed by western blotting. (B) CHOP knockdown. 22Rv1 cells were treated with control or CHOP siRNAs for 24 hours and then with 0.25 μM DOX and 2.5 μM gamitrinib for 24 hours as indicated. The cell viability was analyzed by MTT assay. Data are mean ± SEM of two independent experiments performed in triplicate. *, p < 0.05; **, p < 0.004. (C) CHOP knockdown and Bim expression. After treatment with control or CHOP-#1 siRNAs, 22Rv1 cells were incubated with DOX and gamitrinib alone or in combination for 24 hours as indicated and cell extracts were analyzed by western blotting. (D) Effect of combination drug treatment after Bim silencing. After treatment with Bim siRNA, 22Rv1 cells were incubated with 0.25 μM DOX and 2.5 μM gamitrinib for 24 hours as indicated and cell viability was analyzed by MTT assay. Data are mean ± SEM of two independent experiments performed in triplicate. *, p < 0.05. (E) Expression of Bcl-2 family proteins and DR5. 22Rv1 cells were treated with 0.25 μM DOX and 2.5 μM gamitrinib for 24 hours as indicated. After extraction and reverse transcription of RNA, the cDNA of interest was amplified by PCR.