Figure 2.

Human cells depleted for TDP1 and APE1 exhibit synergistic hypersensitivity to MMS. (a) WT or TDP1^KD MRC5 cells were subjected to control non-targeting siRNA ‘Mock’ or siRNA against human APE1. Cell extract was fractionated by SDS–PAGE and analyzed by immunoblotting using anti-APE1 antibodies (Novus). (b) Control human MRC5 ‘WT’, TDP1^KD, APE1^KD or cells depleted for both APE1 and TDP1 ‘TDP1^KD APE1^KD’ were incubated with the indicated concentrations of MMS and percentage survival calculated from three independent replicates. Data are the mean ± s.e.m. Asterisks denote statistical difference (P < 0.01, t-test) between APE1^KD and TDP1^KD APE1^KD cells (c) WT and TDP^KD cells were incubated with DMSO or 150 μM of the APE1 inhibitor CRT0044876 ‘APEi’ for 2 h followed by an additional incubation with the indicated concentrations of MMS for 15 min at 37°C. Cell survival was calculated from the average of three independent experiments ± s.e.m. Asterisks denote statistical difference (P < 0.02; t-test) between mock and APEi-treated TDP1^KD cells (d) MRC5 cells were incubated with the indicated doses of MMS for 15 min at 37°C and DNA SSBs and alkali labile sites quantified by alkaline comet assays. Average tail moment from 50 cells/sample was measured using Comet Assay IV software (Perceptive) and data are the average ± s.e.m. of three independent experiments. Asterisks denote statistical difference (P < 0.05; t-test) between APE1^KD and TDP1^KD APE1^KD cells.