Fig. 5.

MCF10A cells expressing D239Y accumulate double strand breaks in S and G2/M. (A) MCF10a pools expressing WT or D239Y NTH1 were treated with 30 mM H₂O₂ for 30 min on ice and allowed to recover for 1, 4 and 8 h. Cells were stained with γH2AX/FITC antibody to assess the levels of double-strand breaks and propidium iodide for cell cycle phase, and analyzed by flow cytometry. Data are plotted as the mean ± SEM (n = 3). Within each cell line, + denotes P < 0.01 and ++ denotes P < 0.0001 comparing 1-h to 4-h and 4-h to 8-h recoveries in each cell-cycle phase. D239Y has a significantly higher percentage of γH2AX-positive cells (P < 0.01) than WT at 1-h, 4-h, and 8-h recovery time and in every phase of the cell cycle, except for the 1-h G0/G1 point where WT is higher (P < 0.01). (B and C) γH2AX (B) and phosphorylated CHK1 (C) Western blots of cells synchronized and treated with 30 mM H₂O₂ and allowed to recover for 1, 4, and 8 h before lysis, as in A. Lanes 1–6 are WT, and lanes 7–12 are D239Y. Lanes 1, 2, 8, and 9 are 1-h recovery time; lanes 3, 4, 9, and 10 are 4-h recovery time; lanes 5, 6, 11, and 12 are 8-h recovery time. Lanes 1, 3, 5, 7, 9, and 11 are untreated controls; lanes 2, 4, 6, 8, 10, and 12 are H₂O₂ treated.