Figure 6. NVP-AUY922 abrogates CHK1-mediated G₂/M arrest in the presence of DNA damage inducing cell cycle disruption in p53 null cell lines only.

The ability of NVP-AUY922 to abrogate G₂ arrest in the presence of dsDNA damage was investigated by western blot and FACS analysis in all four cell lines (p53 status: HCT116 p53wt, Hela HPV-positive, HN5 and HN3 p53 mutant). (A) HeLa and HN3 cells were pre-treated with NVP-AUY922 for 16 h at the concentrations indicated. Cells were then mock irradiated or irradiated with 4 Gy and 4 h later whole cell lysates harvested, 30 µg protein resolved on 10% SDS-PAGE then probed for phospho-H2ax, phospho-histone H3 and phospho-CHK1 by western blot. (B) The effect of NVP-AUY922 on depletion of total-CHK1 was also investigated in unirradiated cells with γ-tubulin probed as loading control. (C+D) Cells were exposed to the NVP-AUY922 concentrations indicated for 16 h before mock irradiation or irradiation with 4 Gy. Cells were fixed 9 h and 48 h post-irradiation before staining for the mitotic marker p-histone H3 and DNA content with propidium iodide. SubG₁ and >4N populations were quantified by FACS analysis. Data represents ± SEM of three independent experiments each recording at least 10,000 events. Statistical analysis carried out by two-tailed t-test between the groups indicated, *p<0.05 **p<0.01 ***p<0.001.