Fig 2: Effect of chromatin density on local chromatin motion in HeLa cells.(A) Cellular DNA contents in indicated HeLa cell populations measured by flow cytometry. Each histogram represents more than 16,000 cells. Asynchronous and +lovastatin data were reproduced from Fig. 3B. (B) Quantification from data in (A). (C) Reconstructed 3D images of HeLa nuclei with indicated conditions. Asynchronous and G1 phase nuclei images were reproduced from Fig. 3D. (D) Quantitative data of nuclear volume (means ± SE) of indicated HeLa cell populations. Asynchronous and G1 data were reproduced from Fig. 3E. Mean volumes: lanes 3 (1093 µm3, n = 29 cells) and 4 (831 µm3, n = 30 cells). ***P < 0.0001 by Wilcoxon rank sum test for lanes 2 versus 3 (P = 1.6 × 10-18), 2 versus 4 (P = 2.0 × 10-12), and 3 versus 4 (P = 3.7 × 10-7). (E) MSD plots (±SD among cells) of the nucleosome motion in HeLa G1 (magenta), replication-inhibited (pale pink), and replication-inhibited + ATMi and ATRi (purple) cells. G1 data were reproduced from Fig. 4A. Nucleosome trajectories used per cell: 617 to 875; n = 15 cells per sample. **P < 0.001 by Kolmogorov-Smirnov test for G1 versus replication-inhibited (P = 3.5 × 10-4). N.S. for G1 versus replication-inhibited + ATMi and ATRi (P = 0.68). (F) Inhibition of Chk1 and Chk2 activities by the inhibitor treatment. Western blotting of cell lysates using indicated antibodies. Asterisks denote positions of phosphorylated Chk1. The bottom values indicate the signal intensity ratio of phosphorylated Chk1 or Chk2 versus Chk1 or Chk2, respectively.

Fig 3: Assessment of the effects of ATM, ATR and DNA-PKcs inhibitors on the activation of G1 checkpoint in irradiated G0 cells of SD 82-6 hTert cell cultures. (a) As in Figure 1b for cells exposed to 0 or 1 Gy in the presence of ATMi as indicated. (b) As in (a) for cells exposed to CHK2 inhibitors as indicated. (c) As in (a) for cells exposed to CHK1 inhibitors as indicated. (d) As in (a) for unirradiated cells treated with DNA-PKcsi in the presence or absence of ATMi and or ATRi. (e) As in (d) for cells exposed to 1 Gy of X-rays. (f) As in panel (a), for cells exposed to 0 or 10 Gy in the presence or absence of a CHK2 inhibitor as indicated. Broken lines show results from (b) without data point to avoid congestion. Plotted is the mean and SE from three independent experiments. See also Table S3 for more information.

Fig 4: DNA damage and DNA damage responses upon H2O2 challenge. a, c, e, g Confocal images showing the basal region of the organ of Corti cultures treated with either culture medium alone (a, e) or containing 0.5 mM H2O2 (c, g) for 5 h before being maintained in culture medium alone for 3 days. The samples were then immunolabeled for myosin 7A (red, a, c, e, g), ?H2AX (green, a and c) and 53BP1 (green, e and g). Scale bars: a, c, e and g = 10 µm. b, d, f, h Higher magnification images of representative OHC and IHC nuclei from all conditions tested. Scale bar = 2.5 µm. i Representative Western blot analysis using antibodies against ?H2AX, 53BP1, DDB2, p-Chk1, p-Chk2, p53, and ß-actin in whole cochlear extracts. j, k Histograms representing the levels of ?H2AX, 53BP1, DDB2, p-Chk1, p-Chk2, and p53 in control and in 0.4 and 0.5 mM H2O2-exposed groups (n = 6 cochleae per condition). ß-Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey’s test (*P = 0.04, **P = 0.01, ***P = 0.001 vs. H2O2 0 mM). All experiments were performed in triplicate

Fig 5: Inhibition of PARP1-induced cell cycle arrest at the G2/M checkpoint. (A) Cell cycle analysis of TE6 and TE9 cells transfected with siPARP1 (n=3, cells transfected with siPARP1 were compared with the NC for each cell cycle). Compared with NC, siPARP1 significantly increased the ratio of cells in the G2/M phase and significantly decreased the ratio of cells in the G0/G1 phase. (B) Schema of ATM-Chk2-cdc25c pathways and cdc2/cyclin B1. (C) Western blot analysis showed that inhibition of PARP1 induced cell cycle arrest at the G2/M checkpoint through the ATM-Chk2-cdc25c pathway. PARP, poly (ADP-ribose) polymerase-1; siPARP1, small interfering RNA against PARP1; NC, negative control; KD, knock-down; cdc, cell division control; ATM, ataxia telangiectasia mutated; Chk2, checkpoint kinase 2.