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. 2016 Sep 5;44(21):10259–10276. doi: 10.1093/nar/gkw791

Figure 2.

Figure 2.

N-OH-PhIP triggers the DNA damage response in a proliferation-dependent manner. (A) Dose-dependent activation of the DDR by N-OH-PhIP in Caco-2 cells, HCT116 cells and human colonic epithelial cells (HCEC) after 14 h. Samples were analyzed by SDS-PAGE and western blotting as indicated. Hsp90 was visualized as loading control. Etoposide (ETO) was used as positive control (B) Time-dependent activation of ATR-CHK1 signaling in Caco-2 and HCT116 cells exposed to N-OH-PhIP or UV-C light. Cells were treated with 5 μM N-OH-PhIP or irradiated with UV-C (20 J/m2) and incubated for up to 24 h. Samples were subject to SDS-PGE followed by Western blot analysis as indicated. (C) Cell cycle distribution of HCT116 cells upon serum deprivation. Cells were maintained in normal growth medium (10% FCS) or in serum deprived medium (0.25% FCS) for 24 h. Cell cyle distribution was determined by flow cytometry. (n = 4); ****P < 0.0001, ***P < 0.001, *P < 0.05 versus control (10% FCS). (D) Effect of cell proliferation on ATR-mediated DDR following N-OH-PhIP. Cells were pre-conditioned in medium with 10 or 0.25% FCS for 10 h, treated with N-OH-PhIP (10 μM) or ETO (5 μM) in the conditioned medium and harvested after 14 h (total 24 h). Cells were analyzed as described above.