ADP-ribosylation of histone variant H2AX promotes base excision repair (2020)
Figure 5

Figure 5. ADP-ribosylation of H2AX suppresses γH2AX.

(A) PARP inhibition facilitates γH2AX following oxidative stress. The cells were pre-treated with olaparib (1 µM, 1 hour) prior to H2O2 stimulation. 0.1 % DMSO in medium for 1 hour as mock. γH2AX or H2AX was detected using Western blotting and immunostaining assays. The intensity of γH2AX levels were examined by Western blotting normalized by β-actin and averaged. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (left panel). The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (right panel). Scale bar, 2 μm.

(B) The effect of H2AX E141 ADP-ribosylation on γH2AX following oxidative stress. γH2AX or H2AX was detected using Western blotting and immunostaining assays. The intensity of γH2AX levels were examined by Western blotting normalized by β-actin and averaged. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (left panel). The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (right panel).

(C) The intensity of γH2AX is increased in cell expressing the E141A mutant following KillerRed-induced oxidative DNA lesions (left panel). Scale bar, 2 μm. The foci were indicated with white arrowheads. Scale bar, 0.5 μm. Right panel: the relative intensity of γH2AX at the site of tetR (intensity of γH2AX at the site of tetR/intensity of γH2AX area away from tetR) was quantified. Mean value with SD is the relative intensity of γH2AX in 20 cells. P-values were calculated using Student's t-test. **P < 0.01.

(D) The level of MDC1 at KillerRed-induced oxidative DNA lesions is increased in the cells expressing the E141A mutant (left panel). Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: the percentage of MDC1 foci co-localized with KR was quantified. Mean value with SD is from 20 cells. P-values were calculated using Student's t-test. ***P < 0.001.

(E) The recruitment of Neil3/MDC1 chimera but not wild type Neil3 to the KR-induced oxidative lesions in cells expressing the E141A mutant (left panel). Neil3/MDC1 chimera: replaced the GRFs of Neil3 with the tandem BRCT domains of MDC1. Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: The percentage of detected protein foci co-localized with KR was quantified. Mean value with SD is from 20 cells. P-values were calculated using Student's t-test. ***P < 0.001.

(F) AP sites were measured in cells expressing the E141A mutant or the Neil3/MDC1 chimera, according to the standard curve. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. N.S.: Non Significant, *P < 0.05 and **P < 0.01.

(G) DNA replication of U2OS cells expression with individual wild type (WT), the H2AX KO + WT, the E141A mutant (E141A) or the Neil3/MDC1 Chimera (Chimera) were analyzed by BrdU incorporation following treatment with H2O2, MMS or mock (PBS). Represented images are shown in the left panel. The graph shows percentages of BrdU-positive cells expressing WT, E141A or Chimera (right panel). Values are mean ± SD of three assays. P-values were calculated using Student's t-test. *P < 0.05 and ***P < 0.001. Scale bar, 30 μm.