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. 2016 Jul 20;7:12235. doi: 10.1038/ncomms12235

Figure 5. The catalytic activity of SIRT7 is required for efficient DSB repair.

Figure 5

(a) NHEJ efficiency was determined by FACS in SIRT7- or Ku80-deficient EJ5-HEK293 cells. Each bar represents the mean±s.d. for triplicate experiments. KD efficiency of SIRT7 and Ku80 was examined by western blotting. (b) HR efficiency was determined by FACS in SIRT7- or BRCA1-deficient DR-GFP-U2OS cells. Each bar represents the mean±s.d. for triplicate experiments. KD efficiency of SIRT7 and BRCA1 was examined by western blotting. (c) Rescue experiments for NHEJ or HR deficiency induced by SIRT7 depletion. EJ5-GFP-HEK293 cells (left) or DR-GFP-U2OS cells (right) stably expressing SIRT7 siRNA-1-resistant SIRT7wt (rSIRT7wt) or siRNA-1-resistant SIRT7H187Y (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 48 h, and collected and analysed by FACS. Each bar represents the mean±s.d. for triplicate experiments. (d) SIRT7 occupancy at chromatin flanking DSB generated by endonuclease I-SceI. DR-GFP-U2OS cells transfected with I-SceI were collected at different time points and subjected to ChIP assay, with antibodies against SIRT7. The final DNA extractions were amplified by quantitative real-time PCR using primer that covers the DNA sequences flanking the I-SceI site. Each bar represents the mean±s.d. for triplicate experiments (left). 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 SIRT7. Each bar represents the mean±s.d. for triplicate experiments (right). (e) The efficiency of SIRT7 KD and overexpression of I-SceI, rSIRT7wt or rSIRT7H187Y in DR-GFP-U2OS cells. Tubulin was analysed as an internal control. (f) The KD specificity of SIRT7 siRNA-1 for FLAG-tagged wild-type SIRT7, siRNA-1-resistant rSIRT7wt or rSIRT7H187Y in DR-GFP-U2OS cells. The haemagglutinin (HA)-tagged I-SceI and tubulin were used as loading controls. **P<0.01 (two-tailed unpaired Student's t-test).