Figure 4.

The effect of PARP inhibition on the level of DNA breaks in cell cycle phase-separated K562 cells. (A) Exponentially growing K562 cells were separated into cell cycle phases and exposed to increasing concentrations of CPT in the presence (open symbols) or absence (solid black symbols) of 0.4 μM rucaprib (R) for 30 min, prior to measurement of total DNA breaks by alkaline comet assay. Graphs showing mean Olive tail moment (OTM)±s.e.m. from four independent experiments expressed as percentage of control untreated cells. (B) Similarly, elutriated (G1, S, and G2) K562 cells were exposed to increasing concentrations of CPT±0.4 μM rucaprib (R) for 30 min, and DNA DSB measured using the neutral comet assay. Data are mean+s.e.m. from single representative experiment expressed as a percentage of control. (C) K562 cells were treated with increasing concentrations of CPT in the presence (black bars) or absence (white bars) of 0.4 μM rucaparib for 30 min, and then separated into individual cell cycle phases and stained for the presence of γH2AX foci. Foci number per cell was measured using ImageJ and the macro PZFociEZ, as described in methods. Data show mean±95% confidence interval calculated from at least 100 cells per treatment, from one experiment (representative of three individual experiments).