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. 2020 Jan 29;9:e51636. doi: 10.7554/eLife.51636

Figure 6. SIRT6 can recruit enzymes of both the NHEJ and HR repair pathways.

(A, B) Percentage repair enzymes that are co-localized with SIRT6-LacR-GFP/Cherry at LacO sites. IF with Flag antibody. Data are averages for 3–6 experiments (with error bars representing SEMs) (*, p<0.05; **, p<0.005; ***, p<0.0005; ****, p<0.00005).

Figure 6.

Figure 6—figure supplement 1. SIRT6 can recruit enzymes of boththeNHEJ and HR repair pathways.

Figure 6—figure supplement 1.

(A) Laser-induced damage imaged by IF 30 min after damage. The images show recruitment of p-ATM, 53BP1 or BRCA1 in shControl or shSIRT6 U2OS.
Figure 6—figure supplement 2. SIRT6 can recruit enzymes of boththeNHEJ and HR repair pathways.

Figure 6—figure supplement 2.

(A, B) Co-localization of 53BP1 and BRCA1 with SIRT6-LacR-GFP (53BP1: n = 247, p<0.005; BRCA1: n = 223, p<0.005) and HY-LacR-GFP (53BP1: n = 113, p<0.005; BRCA1: n = 122, p<0.005), compared with GFP-LacR (53BP1: n = 130; BRCA1: n = 157). The data shown in panel (B) are averages from 3–5 experiments (error bars are SEMs). (C, D) Comparison of co-localization of endogenous 53BP1 (n = 247) and BRCA1 (n = 223) and transfected 53BP1-Flag (n = 111) and BRCA1-Flag (n = 146) with SIRT6-LacR-GFP, assessed by IF.
Figure 6—figure supplement 3. SIRT6 can recruit enzymes of boththeNHEJ and HR repair pathways.

Figure 6—figure supplement 3.

(A) Schematic representation of the Tethering assay with inhibition of signaling using Wortmannin. (B–D) Co-localization of γH2AX (B), 53BP1 (C), and BRAC1 (D) with SIRT6-LacR-GFP or GFP/Cherry-LacR after 24 hr with or without Wortmannin. Data are averages for three experiments (error bars are SEMs). (B) Co-localization with γH2AX (0 μM: n[SIRT6] = 43, n[GFP] = 41; 1 μM: (n[SIRT6] = 42, n[GFP] = 42; 10 μM: (n[SIRT6] = 42, n[GFP]=41). (C) Co-localization with 53BP1 (0 μM: n[SIRT6] = 32, n[GFP] = 32; 1 μM: n[SIRT6] = 30, n[GFP] = 32; 10 μM: n[SIRT6] = 33, n[GFP] = 31). (D) Co-localization with BRCA1 (0 μM: n[SIRT6] = 30, n[GFP] = 32; 1 μM: n[SIRT6] = 31, n[GFP] = 31; 10 μM: n[SIRT6] = 32, n[GFP] = 31). (E) Co-localization of Ku80-Flag or MRE11-Flag with SIRT6-LacR-GFP or GFP/Cherry-LacR after 24 hr with or without Wortmannin. Data are averages for three experiments (error bars are SEMs). For Ku80-Flag — 0 μM: n[SIRT6] = 45, n[Cherry] = 53; 10 μM: n[SIRT6] = 45, n[Cherry] = 58. For MRE11-Flag — 0 μM: n[SIRT6] = 50, n[Cherry] = 56; 10 μM: n[SIRT6] = 60, n[Cherry] = 51. *, p<0.05; **, p<0.005; ***, p<0.0005; ****, p<0.00005).