Extended Data Fig. 2: Delays in mitosis do not induce DNA damage in subsequent G1 cells.
From: MDM2 functions as a timer reporting the length of mitosis
a, Asynchronous (AS) hTERT-RPE1 p53WT cell cultures were plated to mimic the conditions in Fig. 1a–c, then 17 h later the replated cells were treated with DMSO (Control), 30 µg/ml bleomycin (Bleo) or 200 ng/ml neocarzinostatin (NCS) for 1 h to induce DNA damage. To test the effect of mitotic arrest on DNA damage, hTERT-RPE1 p53WT cells were treated with 25 ng/ml nocodazole for 18 h. Mitotic cells harvested by shake-off and nocodazole washed-out, replated, and then 17 h later treated with DMSO, Bleo or NCS for 1 h. In both cases, DNA damage induction was assessed by Western blotting (n = 3 independent experiments with similar results). b,c, Graphs calculated from a showing the quantification of phosphorylated p53 (p53 pS15) (b) and Chk2 (Chk2 pT68) (c) relative to total proteins (mean ± s.d.; n = 3 independent experiments; p53 pS15/total, p = 2.4E−8 AS Control vs. AS Bleo, p = 2.0E−9 AS Control vs. AS NCS, p = 4.2E−7 M-G1 Control vs. M-G1 Bleo, p = 9.5E−8 M-G1 Control vs. M-G1 NCS, p = 0.99 M-G1 Control vs. AS MD-224, p = 0.28 AS Control vs. AS MD-224; Chk2 pT68/total, p = 4.2E−5 AS Control vs. AS Bleo, p = 1.0E−4 AS Control vs. AS NCS, p = 9.5E−4 M-G1 Control vs. M-G1 Bleo, p = 5.0E−3 M-G1 Control vs. M-G1 NCS, p = 0.99 M-G1 Control vs. AS MD-224, p = 0.99 AS Control vs. AS MD-224). d, hTERT-RPE1 p53WT FUCCI cells treated as in a were stained for 53BP1. The FUCCI marker was directly visualised using fluorescence microscopy. e, Scatter plots of the number of 53BP1 foci in G1 cells from d, showing individual cells with the mean values from 3 independent experiments (mean ± s.d.; p = 0.99 AS Control vs. M-G1 Control, p = 0.93 AS Bleomycin vs. M-G1 Bleomycin, p = 0.25 AS NCS vs. M-G1 NCS). Data were analysed using one-way ANOVA test with Tukey’s multiple comparisons (b,c) and two-way ANOVA test with Å Ãdák’s multiple comparisons test (e) (ns, not significant; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Source numerical data and unprocessed blots are available in source data.
