Figure 6: SIRT7 desuccinylates H3K122succ at DSB sites.

(a) MCF-7 cells were treated with 40 nM VP16 or 1 μM CPT for 8 h. Histones were extracted for western blotting analysis with the indicated antibodies. (b) Control or SIRT7-depleted MCF-7 cells (left) or U2OS cells (right) were treated with 1 μM CPT for 8 h followed by histone extraction and western blotting analysis with the indicated antibodies. The efficiency of SIRT7 KD and DNA-damage effect induced by CPT were monitored by western blotting of whole-cell lysate using antibodies against SIRT7 and γH2AX, respectively. (c) Control or SIRT7-depleted U2OS cells were exposed to 10 Gy of IR and collected at different time points for histone extraction and western blotting analysis with the indicated antibodies. The efficiency of SIRT7 KD and IR treatment was monitored by western blotting of whole-cell lysate using antibodies against SIRT7 and γH2AX, respectively. (d) Control or SIRT7-depleted U2OS cells were subjected to laser microirradiation and immunofluorescent analysis of H3K122succ and γH2AX at 5 min after microirradiation. Scale bar, 10 μm. (e) U2OS cells transfected with control siRNA or siPARP1 were subjected to laser microirradiation and immunofluorescent analysis of H3K122succ and γH2AX at 5 min after microirradiation. Scale bar, 10 μm. (f) H3K122succ levels in e,f were measured at and beside damage sites using ImageJ. At least 30 independent cells were scored. Data are represented as mean±s.e.m. **P<0.01 (two-tailed Student’s t-test). (g) Control or DR-GFP-U2OS cells stably expressing rSIRT7wt or rSIRT7H187Y were transfected with control siRNA or siSIRT7-1 as indicated. Twenty four hours later, the cells were transfected with pcDNA3.1 vector or I-SceI for 40 h and subjected to qChIP analysis with antibodies against H3K122succ. Each bar represents the mean±s.d. for triplicate experiments. **P<0.01 (two-tailed unpaired Student’s t-test).