Fig 2: Effect of RYBP on the synergy between PARP inhibitor and ATM inhibitor. (A). In control U2OS cells, addition of ATM inhibitor (KU-55933) dramatically reduced the IC50 of PARP inhibitor (ABT-888) from 220 µM to 26 µM. (B). In RYBP-expressing cells, addition of ATM inhibitor (KU-55933) slightly reduced the IC50 of PARP inhibitor (ABT-888) from 83 µM.

Fig 3: Double-strand break detection is influenced by FGF1 expression.a MTT assays were used to compare viability of A2780, A2780DPP and A2780DPP cells treated with 10 µM KU55933 (ATMi) or 2 µM NU7026 (DNA-PKi) in response to cisplatin (0–25.33 µM). b qRT-PCR analysis was used to assess ATM expression relative to 18S rRNA in response to 5 nM, 10 nM and 20 nM ATM siRNA (±compound SD). c MTT assays were used to compare the viability of A2780, A2780DPP and A2780DPP cells transfected with 20 nM siRNA scrambled negative control or 20 nM siATM in response to cisplatin (0–25.33 µM). Representative immunoblot analysis comparing the phosphorylation of ATM (S1981) over indicated timepoints following 3 h treatment with 3 µM cisplatin in thymidine synchronised cells (2 mM, 24 h) in d A2780, e A2780DPP and f A2780DPP FGF1KD cells. A timecourse of FGF1-dependency H2AX (S139; ?H2AX) phosphorylation was investigated before (g–i) and following treatment with 10 µM KU55933 (ATMi) (j–l) in A2780, A2780DPP and A2780DPP FGF1KD cells. Results are illustrative of four repeat experiments. Pairwise comparisons of mean IC50 values and relative gene expression were calculated by Student’s t-tests. Scale bar = 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001. Ns not significant.

Fig 4: Effect of RYBP expression on phosphorylation of ATM after DNA damage. (A). Left; cells transfected with GFP-RYBP and treated with calicheamicin (50 pM, 1 h). Arrows indicate that cells expressing GFP-RYBP have less p-ATM foci. Right; quantification of p-ATM foci per nucleus (at least 50 nuclei from 3 independent experiments) in control and RYBP-expressing cells. (B). Left; cells transfected with GFP-RYBP and treated with camptothecin (10 µm, 1 h). Arrows indicate that cells expressing GFP-RYBP have less p-ATM foci. Right; quantification of p-ATM foci per nucleus (at least 50 nuclei from 3 independent experiments) in control and RYBP-expressing cells. * p < 0.5 (Student’s t-test). In the merged images, GFP-RYBP (green) and p-ATM (red). Scale bar is 10 μm.

Fig 5: Resveratrol induces S phase arrest, DDR and cellular senescence. (A) Cell viability determined by MTT assay. U2OS cells were treated with the indicated concentrations of resveratrol (RSV) for 48 h, and cell viability was determined by MTT. **P < 0.01, when compared with control group. (B) RSV induces S-phase arrest of U2OS cells. Cells were treated with different concentration of RSV for 24 h and then cell cycle distribution was measured by FACS analysis. Data are presented as the mean ± SD of values from triplicate experiments. (C) Detection of ?-H2AX by immunofluorescence. Cells were treated with the indicated concentrations of RSV for 24 h, stained with mouse anti-?-H2AX(Ser139) antibody and goat anti-mouse TRITC-conjugated secondary antibody, then counterstained with DAPI (blue). (D) Western blotting analysis of p-ATM, p-Chk1 protein levels in U2OS cells treated with different concentration of resveratrol for 24 h. (E) Induction of cellular senescence by RSV. U2OS cells were treated without or with different concentration of RSV for 7 days. Cellular senescence was examined by SA-ß-gal staining. (F) Cell cycle distribution of U2OS cells after being treated with different concentration of RSV for 5 days.