Figure 4.

Depletion of Top1 protects human cells from camptothecin-induced cell death. (a) Control ‘WT’ or TDP1^KD human MRC5 cells were subjected to lentivirus particles containing control non-targeting shRNA or shRNA against human Top1 ‘Top1^KD’ to generate stable cells in which TDP1 or Top1 was depleted separately or together. Cell lysates were fractionated by SDS–PAGE and analyzed by immunoblotting using anti-TDP1 or anti-Top1 antibodies. (b) The indicated cell lines were incubated with 20 μM CPT for 1 h at 37°C and DNA strand breakage quantified using alkaline comet assays. Average tail moments from 50 cells/sample were measured, and data are the average ± s.e.m. of three independent experiments. (c) The indicated MRC5 cells were incubated with increasing concentrations of CPT for 60 min at 37°C and survival was determined from three biological replicates and presented as mean ± s.e.m. (d) Cells were additionally subjected to control siRNA or Top1 siRNA to deplete residual levels of Top1 in TDP1^KD/Top1^KD cells and cell lysate analyzed by SDS–PAGE and immunoblotting. (e) Control ‘WT’, TDP1^KD, Top1^KD or TDP1^KD/Top1^KD cells in which Top1 level was additionally depleted by siRNA were treated with increasing concentrations of CPT for 60 min at 37°C and survival determined from three biological replicates and presented as mean ± s.e.m. Asterisks denote statistical difference (P < 0.05; t-test) between TDP1^KD and TDP1^KD Top1^KD cells.