Figure 5. RECQ1-depleted cells repair H₂O₂-induced DNA damage in PARP activity-dependent manner.

A. RECQ1-depleted cells are sensitive to H₂O₂ exposure. Cells transfected with control or RECQ1 siRNA were exposed in quadruplicate to increasing doses of H₂O₂ for 10 min followed by growth in complete medium and their survival was measured 72 h later by MTS assay. Percentage of control growth was plotted for each data point, representing the mean ± SD of three independent experiments. Depletion of RECQ1 is shown by Western blot. GAPDH serves as loading control. B. Total DNA strand breaks in control or RECQ1-depleted cells. Alkaline Comet Assay was used to measure the total DNA strand breaks with or without prior exposure to PARP inhibitor ANI (100 µM, 16 h) in control or RECQ1-depleted cells that were either untreated or allowed to recover for indicated time following treatment with H₂O₂ (10 µM, 5 min). The total DNA strand breaks were quantitated as % tail DNA representing the mean of three independent experiments with SD indicated by error bars. At least 100 cells were analyzed in each experiment. C. Colony formation of H₂O₂-treated control or RECQ1-depleted cells grown in the presence or absence of ANI. Cells were incubated with or without ANI (10 µM, 16 h) before treatment with H₂O₂ (1 mM, 10 min) or left untreated. Following removal of H₂O₂, cells were grown in the presence or absence of ANI (10 µM) in complete medium and colonies were stained and counted after 14 days. As reported previously, depletion of RECQ1 resulted in reduced colony formation [3]. Results are expressed as the relative percentage of colonies as compared to untreated controls for each siRNA following adjustment for plating efficiency using untreated plates and are the means of duplicate experiments, each performed in triplicate. Ctl, control.