Fig 2: γH2AX expression is increased in E13.5 Polμ−/− retinas.(a) Active DNA repair was evaluated by Western blot in WT and Polμ−/− retinal protein extracts. γH2AX levels were compared with those of H2AX and H2AII. (b,c) γH2AX immunostaining (green) showed a focal distribution in WT and Polμ−/− retinas. (d) The density of γH2AX-positive cells was quantified in whole-mount retinas. (e) The number of γH2AX-positive foci per positive cell was also scored in dissociated retinal cells (n > 100). Histogram shows the mean ± standard error of mean (s.e.m.), as well as the individual values in (d). *P < 0.05 and ***P < 0.001 vs. corresponding controls.

Fig 3: DLGAP1-AS2 upregulated CD151 by interacting with E2F1 to regulate the radiation resistance of rectal cancer stem cells.(A–B) Western blot for measuring the protein levels of CD133, MDR1, BCRP1 and γ-H2AX and AKT/mTOR/cyclinD1 signaling. *P<0.05, **P<0.01, ***P<0.001. The error bar represents the mean ± SD. Each experiment was repeated three times.

Fig 4: Radioprotective effects of cinobufagin. (A) IM-9 and HuT 78 cells were treated with or without 50 nM of cinobufagin for 1 h, then irradiated with the indicated dose of radiation and incubated for 24 h. The expression levels of ATM, CHK2, p53 and H2AX phosphorylation were evaluated using western blotting. (B) IM-9 and HuT 78 cells were treated with or without cinobufagin (25 or 50 nM) for 1 h prior to irradiation with the indicated radiation doses for 24 h. The cells were analyzed for Annexin V/PI staining using flow cytometry. Representative bar graphs show the mean ± SEM of three independent experiments; **P<0.01 and ***P<0.001 compared with the control, +P<0.05 and ++P<0.01 compared with the irradiated control. (C) Cinobufagin or the vehicle (1.1% DMSO in PBS) were administrated intraperitoneally 24 h prior to total body radiation (8 Gy) on C57BL/6 mice (n=8 mice/group) and survival was observed for 15 days after exposure. (D) Cinobufagin or the vehicle were administrated intraperitoneally 24 h prior to total body radiation (3 Gy) on C57BL/6 mice (n=5 mice/group). The spleen (left panel) and BM (right panel) were harvested at 24 h following radiation, and the number of splenocytes and BM cells were counted by Trypan blue exclusion. Data shown are the mean ± SEM. Statistical significance was represented for each control group (**P<0.01, ***P<0.001, ****P<0.0001). ns, not significat; IR, ionizing radiation; TBI, total body irradiation; ATM, ataxia telangiectasia mutated; CHK2, checkpoint kinase 2; H2AX, H2A histone family member X.

Fig 5: Rescue of 53BP1 IRIF in RNF8-/- MEFs. (A)RNF8-/- MEFs transiently expressing the indicated FLAG-tagged H2AX proteins were exposed to IR (9 Gy) and 4 h later processed for immunofluorescence. More than one hundred cells with high level of FLAG signal were scored for 53BP1 IRIF. The percentages of cells with more than 10 53BP1 foci (means ± 1 SD) from 3 to 4 independent experiments are indicated. Scale bar = 10 μm. K1315R, K13R/K15R double substitution. (B)RNF8-/- MEFs transiently expressing the indicated FLAG-tagged H2AX proteins were exposed to IR (9 Gy) and 4 h later processed for immunofluorescence using antibodies reacting with conjugated ubiquitin (FK2) or K63-linked polyubiquitin chains (K63). (C)RNF8-/- MEFs transiently expressing GFP-tagged RNF8 were exposed to IR (9 Gy) and 4 h later processed for immunofluorescence using antibodies reacting with GFP, conjugated ubiquitin (FK2) or K63-linked polyubiquitin chains (K63).