Fig 1: SIRT3 mediates cGAS decrotonylation.A, IB analysis was performed on WCLs and anti-Flag IPs of HeLa cells transfected with the Flag-cGAS plasmid and pretreated with 1 μM TSA or 10 mM NAM for 10 h to observe the effects on cGAS crotonylation levels. B, IB analysis was performed on WCLs and anti-GFP IPs of HeLa cells transfected with the indicated constructs. C, IB analysis was performed on WCLs and anti-Flag IPs of HeLa cells transfected with increasing amounts of Myc-SIRT3 plasmid (1 μg, 2 μg, or 4 μg). D, IB analysis was performed on WCLs and anti-Flag IPs of HeLa cells transfected with the indicated siRNAs and plasmids. E, Co-IP of endogenous SIRT3 with Flag-cGAS from HeLa cells at the indicated times post-IR (8 Gy γ-ray). F, in vitro cell-free reaction assay of cGAS crotonylation and decrotonylation by CBP and SIRT3, respectively. Workflow of the in vitro assay (upper panel). The samples were analyzed by IB. cGAS, cyclic GMP-AMP synthase; IB, immunoblotting; IP, immunoprecipitate; NAM, nicotinamide; TSA, trichostatin A; WCLs, whole-cell lysates.
Fig 2: SIRT3 deficiency impairs DNA repair and prevents HR repair.A, survival of HeLa cells in different groups after 0, 2, 4, and 8 Gy irradiation was detected by colony formation assay. B, HeLa cells were transfected with the indicated siRNAs, and later formation assays were conducted. The data are presented as the means ± SDs of triplicate experiments. ∗p < 0.05, ∗∗p < 0.01. C and D, sensitivities of SIRT3-depleted HeLa cells (C) and MCF-7 cells (D) to DNA damage or replication stress–inducing agents were determined by MTS assays. The data are shown as the means ± SDs of biological triplicates. ∗p < 0.05, ∗∗p < 0.01. E and F, HeLa cells were transfected with the indicated SIRT3 siRNAs. After 24 h, cells were treated with IR (8 Gy) for 1 h. An immunofluorescence assay was performed to test the extent of RAD51 foci formation using the corresponding antibodies. Scale bar, 10 μm. The data are presented as the means ± SDs of triplicate experiments. ∗∗p < 0.01. G, flow cytometry analysis of HR repair efficiency in cells expressing different cGAS variants. H, DR-GFP reporter assay was performed to examine the HR efficiency, as described in the “Experimental procedures” section. The represented data represented are shown as the means ± SDs of triplicate experiments. ∗∗p < 0.01. cGAS, cyclic GMP-AMP synthase; CPT, camptothecin; ETO, etoposide; HR, homologous recombination; HU, hydroxyurea; IR, ionizing radiation; MMC, mitomycin C; ns, nonsignificant.
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