Differential cellular responses to MNNG treatment in TK6 and MT1. (A) Flow cytometric analysis of cell cycle progression. TK6 and MT1 cells were treated with 0.5 μM MNNG for 4 h, continuously cultured in fresh media without MNNG, and harvested for flow cytometry analysis at the indicated time points after MNNG treatment. (B) DNA fragmentation analysis. TK6 and MT1 cells were treated with 0.5 μM MNNG for 4 h, continuously cultured in fresh media without MNNG for 72 h, harvested and extracted for genome to analyze with DNA fragmentation experiment. (C) Detection of phosphorylation level of ATR/ATM kinase substrates at 4 h time point after 0.5 μM MNNG treatment. TK6 and MT1 cells were incubated in the presence or absence of 0.5 μM MNNG for 4 h. Whole cell lysates were analyzed by western blotting with an anti-phospho-(Ser/Thr) ATM/ATR substrate antibody. This antibody preferentially detects endogenous levels of proteins containing the ATM/ATR substrate motif. Equivalent gel loading was confirmed by probing with an antibody against β-Actin.