Quantifying DSBs and p53 dynamics in individual living cells. (A) Schematic drawing of the 53BP1, p53 and H2B reporters. (B) Time-lapse images of MCF7 cells expressing the reporters in (A) after damage (10Gy γ-irradiation) at the indicated time points. The 53BP1-mCherry images are maximum projections of z-stacks through the nucleus (see Methods section). (C) Quantification of the number of 53BP1-mCherry foci (red) and the normalized average nuclear p53-Venus intensity (blue). Trajectories from three individual cells treated with 5Gy or 10Gy γ-irradiation as indicated are shown. The left panel corresponds to the cell shown in (B). (D) The correlation between the initial numbers of breaks post 5Gy and the number of p53 pulses during the response was analyzed. The median number of DSBs is significantly higher in cells that pulse four times or more compared to cells pulsing only once or twice, but the distributions are overlapping. Red lines indicate the median; p-values were calculated by the Wilcoxon-Mann–Whitney test. Number of cells = 97 (E-F) The correlation of DNA damage after a p53 pulse and the induction of a subsequent pulse was analyzed as indicated (E). The number of breaks after the first p53 pulse is shown for individual cells, binned for the presence or absence of a subsequent pulse (F). The medians are significantly different, but distributions overlap. For the analysis, data sets from cells treated with 5Gy or 10Gy γ-irradiation were combined. Red lines indicate the median; the p-value was calculated by the Wilcoxon-Mann–Whitney test. Number of cells >160. DSBs, double strand breaks.
Loewer et al. BMC Biology 2013 11:114 doi:10.1186/1741-7007-11-114