Figure 8.

Overexpressed FancA rescues the cytotoxicity of combined carboplatin and ganetespib. (a) Left: Ovcar-5 cells were transfected with a control plasmid (pcDNA3) or an expression plasmid for FancA (pMMP_FancA), followed by treatment with ganetespib and/or carboplatin. In all cases, FancA overexpression strongly increased cell viability (P<0.001). Right: Combination Index (CI) plotted against the fraction affected (Fa) for control and FancA-overexpressing Ovcar-5 cells. FancA overexpression abolishes the synergistic action of the combined drugs. (b) FancA overexpression overcomes γH2AX and pH3 accumulation in combination-treated cells (compare the last three lanes). Ovcar-5 cells were transfected with a control (pcDNA3) or expression plasmids for 3-myc-tagged FancA (pcDNA_FancA) or non-tagged FancA (pMMP_FancA). Immunoblot analysis performed 24 h after treatment with 20 nM ganetespib and 90 μM carboplatin, alone or in combination. (c) Control and FancA-overexpressing Ovcar-5 and EFO-21 cells were treated for 48 h with the IC50s of ganetespib and/or carboplatin (drug concentrations, cf. Supplementary Table S1) in the presence of 20 μM zVAD. The cells were processed as in Figure 4a and representative images are shown. The extent of chromosome fragmentation per cell was analyzed by counting the fragments in 10 randomly chosen metaphases. Significance was determined by the Student's t-test and the P-value is shown by asterisks (***P-value<0.001)