Figure 1: Cells enter mitosis with variable numbers of γH2AX foci after DNA damage.

(a) The experimental protocol. U2OS cells were synchronized in G2 by thymidine block before exposure to DNA damage (either a brief pulse of etoposide or ionizing radiation, as indicated in the text). Synchronized, damaged cells were treated with nocodazole 30 min later to trap in mitosis cells that had recovered from G2 arrest, before staining for phospho-histone H3 (pH3) and γH2AX. (b) The bar chart plots the average level of checkpoint activation signal, denoted by γH2AX foci number, in U2OS cells that had recovered from G2 arrest and entered mitosis at different time points after etoposide exposure. The average number of mitotic γH2AX foci in undamaged cells arrested in nocodazole for 10 h is shown as control. (c) The chart plots the average percentage of U2OS cells that entered mitosis at different time points after etoposide exposure. (d) The bar chart plots the average level of checkpoint activation signal, denoted by γH2AX foci number, in U2OS cells that had recovered from G2 arrest and entered mitosis at different time points after 1.5 Gy IR treatment. The average number of mitotic γH2AX foci in undamaged cells arrested in nocodazole for 10 h is shown as a control. (e) The chart plots the average percentage of U2OS cells that entered mitosis at different time points after 1.5 Gy IR treatment. (f) RPE1 cells synchronized by thymidine block were exposed to 1 Gy IR, and treated as in a. The bar chart shows the average level of checkpoint activation signal (γH2AX foci) in cells that had recovered from G2 arrest and entered mitosis at different time points after damage exposure. The average number of mitotic γH2AX foci in undamaged cells arrested in nocodazole for 12 h is shown as a control. (g) The chart plots the average percentage of RPE1 cells that entered mitosis at different time points after 1 Gy IR treatment. The experiments in all panels were run in triplicate. Charts depict the mean and s.e.m.