Figure 2: Cells enter mitosis with variable numbers of MDC1 foci after DNA damage in live cell imaging. | Nature Communications

Figure 2: Cells enter mitosis with variable numbers of MDC1 foci after DNA damage in live cell imaging.

From: Homeostatic control of polo-like kinase-1 engenders non-genetic heterogeneity in G2 checkpoint fidelity and timing

Figure 2

(a) The experimental protocol. U2OS cells were synchronized in G2 by thymidine block before exposure to a brief pulse of etoposide. Synchronized, damaged cells were subjected to live cell imaging to monitor damage foci. (b) Serial imaging of U2OS cells expressing MDC1-GFP following exposure to etoposide. Serial images were analysed to identify the first image exhibiting visible dissipation of nuclear fluorescence marking absence of the nuclear envelope (final panel, 11.5 h). MDC1-GFP foci number was enumerated just before nuclear envelope breakdown (NEBD) at mitotic entry, 15 min before this event (penultimate panel, 11.25 h). Experimental procedures were as described in a. Images are representative of multiple independent experiments. Scale bar, 5 μm. (c) The graph depicts the level of checkpoint activation signal, denoted by MDC1 foci number (y axis), retained in cells just before NEBD at mitotic entry, against the time taken to reach NEBD after damage exposure (x axis). The grey bars show the distribution of average retained foci number in cells undergoing NEBD each hour and the black bars show the total number of cells undergo NEBD each hour. (d) Cells depicted in c were grouped into three categories based on the time taken to enter mitosis after DNA damage. Cells that entered mitosis early (1st quartile, red) were compared with those entering around the median (2nd and 3rd quartiles, yellow) or late (4th quartile, green). The early and late categories were selected to include equal cell numbers. (e) Displays the statistical significance of differences in the foci number between the three categories using the analysis of variance/Bonferroni test. Notably, the remaining level of MDC1 foci number at mitotic entry in the three categories of damage-exposed cells is also significantly higher than that from cells without DNA damage treatment (white). The mean (bar) and s.e.m. (whiskers) are shown (***P<0.0001; ****P<0.00001).

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