Figure 4.

ATR inhibition potentiates DSB induction and activates ATM signaling in response to N-OH-PhIP. (A and B) Impact of ATR inhibition on N-OH-PhIP-induced DDR. Caco-2 cells were incubated with N-OH-PhIP (10 μM) in the absence or presence of the ATR inhibitor (ATRi) VE821 for 14 h. Samples were then subjected to SDS-PAGE followed by western blot analysis as indicated. Hsp90 served as loading control. (C) ATR inhibition in human colonic epithelial cells (HCEC) exposed to N-OH-PhIP. Cells were treated and analyzed as described above. (D) DSB induction upon ATR inhibition. Caco-2 cells were treated as stated above and DSB formation was measured using the neutral Comet assay (n = 4); ns: not significant. *P < 0.05, ***P < 0.001. (E) Influence of ATR inhibition on threonine-21 RPA phosphorylation. Caco-2 cells were treated as described in (A) and analyzed for pRPA. Hsp90 was used as loading control. (F) Confocal microscopy of RPA Thr21 phosphorylation. Caco-2 cells were incubated for 14 h as described above, fixed and stained with a p-Thr21-RPA antibody followed by an Alexa 488-coupled secondary antibody (green). Nuclei were counterstained with TO-PRO-3 (blue). Samples were then analyzed by confocal microscopy and processed by ImageJ software. The scale bar represents 5 μm. (G) Quantitative evaluation of pRPA staining. The number of pRPA foci per nucleus were assessed by ImageJ software and evaluated with GraphPad Prism 5.0 (>100 cells per experiment; n = 2).