DNA double strand breaks (DSBs) measured by detection of γH2AX induction in late PD HMECs by mammogram X-rays last longer than in early PD HMECs.
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A. Time-dependence of γH2AX foci formation and disappearance measured by direct microscopic visualization. The foci were counted in 1000 cells per each time and cell subpopulation group. Error bars signify standard error. Simple asterisk (*) refers to statistically significant difference p<0.05 and double asterisk (**) refers to highly significant difference p<0.0001. Mann Whitney test was performed in all samples. B. Time-dependent flow cytometric bivariant detection of propidium iodide (PI)-and γH2AX. Histogram showing the kinetics of γH2AX appearance and disappearance measured by flow cytometry. For early PD HMECs (green), the maximum percentage of γH2AX positive G1-stage cells was reached at 30 minutes after irradiation, whereas in vitro aged HMECs (red) do not reach maximum until 90 min time-point. A minimum of 10,000 cells and two replicas were analyzed by bivariate flow cytometry for each time-point and cell subpopulation. C. Representative dot-plots illustrate that γH2AX labeling lasts longer in the in vitro aged epithelial cells (red dots indicate positive and blue negative γH2AX labeled cells). Cell cycle is displayed using contour plots and γH2AX labeling using pseudo-color plots. Gating set at 3% increment over unspecific background fluorescence for both control and irradiated HMECs. Percentages of positive γH2AX cells at the G1 cell cycle stage are indicated in each dot-plot. For cytometric analysis, early and late PD HMECs were irradiated with 1Gy γ-rays. D. Mean incidence of micronuclei per cell in early and late PD HMECs derived from three different donors and exposed to 0, 2 and 10 automatic X-ray shots under a mammogram device (24 h after irradiation). The micronuclei were counted in 2000 (donor 1) and 500 (donor 2 and 3) binucleates per group. Error bars signify standard error. Asterisk denotes statistically significant difference in a group of irradiated HMECs compared to the shamirradiated controls of each cell subpopulation (Mann Whitney test). Simple asterisk (*) refers to statistically significant difference p<0.05 and double asterisk (**) refers to highly significant difference p<0.0001.