1. Sirt6 Promotes DNA End Joining in iPSCs Derived from Old Mice
Wen Chen, Nana Liu, Hongxia Zhang, Haiping Zhang, Jing Qiao, Wenwen Jia, Songcheng Zhu, Zhiyong Mao, Jiuhong Kang 
Cell Reports 
Volume 18, Issue 12, Pages (March 2017)
DOI: /j.celrep
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2. Cell Reports 2017 18, 2880-2892DOI: (10.1016/j.celrep.2017.02.082)
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3. Figure 1 iPSC Genomic Stability Is Influenced by the Age of the Mice
(A) Representative alkaline comet assays of young and old mouse-derived iPSCs.
(B) Comparison of genomic instability measured by alkaline comet assay between the two groups. The 15 young mouse-derived iPSC lines and 15 old mouse-derived iPSC lines (5 × 5 mice) were analyzed at passages 7–10. We performed the comet assay with iPSC lines with no induction of DNA damage. For each cell line, the tail moments of at least 50 cells were quantified using Cometscore software.
(C) The percentage of DNA content in tails of 15 young and 15 old mouse iPSC lines.
(D) NHEJ efficiency was measured in young and old mouse-derived iPSCs.
(E) HR efficiency was measured in two group cells.
Bar graphs represent mean ± SEM. p Values are according to Student’s t tests in (B)–(E). See also Figures S1 and S2.
Cell Reports  , DOI: ( /j.celrep )
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4. Figure 2
Sirt6−/− iPS Cells Show Genomic Instability and Reduction of NHEJ Efficiency
(A) Western blot analysis of Sirt6, c-NHEJ (Ku70, Ku80, Artemis, XLF, Lig4, and DNA-PKcs), and alt-NHEJ factors (PARP1and Lig3) in young and old mouse-derived iPSCs.
(B) Statistical comparison of Sirt6 protein expression between the two groups. Western blot results were analyzed with Gel-Pro Analyzer 4.0 software.
(C) Representative alkaline comet assays of the OSK and OSK+Sirt6 iPSC lines.
(D and E) Tail moment (D) and percentage of DNA content in tails (E) of six OSK and six OSK+Sirt6 iPSC lines.
(F) Representative alkaline comet assays of the Sirt6+/+ and Sirt6−/− iPSC lines.
(G and H) Tail moment (G) and percent of DNA in tails (H) of six Sirt6+/+ and eight Sirt6−/− iPSC lines.
(I and J) NHEJ efficiency (I) and HR efficiency (J) were measured in Sirt6+/+ and Sirt6−/− iPSCs.
The cell lines in (C)-(H) were analyzed at passages 7–10 and at least 50 cells were quantified for each cell line. Bar graphs represent mean ± SEM. p Values are according to Student’s t tests in (B), (D), (E), and (G)–(J). See also Figures S2–S4.
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5. Figure 3 Sirt6 Directly Interacts with Ku80
(A) FLAG-control or FLAG-Sirt6 viruses were introduced into Sirt6+/+ iPSC clones. Cells were irradiated and immunoprecipitated 30 min after IR exposure (8 Gy). IP was performed using FLAG beads, and FLAG-tagged luciferase was used as a control. Membranes were immunoblotted for Ku80 and FLAG.
(B) Endogenous interaction between Ku80 and Sirt6 in iPSCs. iPSCs were treated with etoposide for 0.5 hr. IP was performed using an anti-Ku80 antibody and normal immunoglobulin G (IgG). Membranes were immunoblotted for Sirt6 and Ku80.
(C) 293T cells were co-transfected with HA-mouse Ku80 and either FLAG-luciferase (Control) or FLAG-mouse Sirt6. IP was performed with anti-HA beads in the absence or presence of EtBr or DNase I. Membranes were immunoblotted for HA and FLAG.
(D) FLAG-control, FLAG-Sirt6, or FLAG-sirt6 mutant (H133Y) viruses were introduced into Sirt6−/− iPSC clones and immunoprecipitated with anti-FLAG beads. Membranes were immunoblotted for Ku80 and FLAG.
(E) Schematic of wild-type mouse Ku80 and the different truncated Ku80 mutants used in this study.
(F) IP of the HA-tagged Ku80 and truncated Ku80 mutants (Ku80-a+b, Ku80-c+d, Ku80-a, Ku80-c, and Ku80-d) in 293T cells.
(G) IP of the different HA-tagged Ku80 variants.
(H) IP of the HA-tagged control, WT Ku80, and Ku80Δ10 that only lacks Aa.
Membranes in (F)–(H) were immunoblotted for Sirt6 and HA. Black arrows indicate specific bands.
See also Figure S5.
Cell Reports  , DOI: ( /j.celrep )
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6. Figure 4
Sirt6-Ku80 Protein Interaction Is Essential for Sirt6 to Improve NHEJ Repair
(A) Representative alkaline comet assays of Ku80+/+ and Ku80−/− iPSCs.
(B and C) Tail moment (B) and percentage of DNA content in tails (C) of five Ku80+/+ and five Ku80−/− iPSC lines.
(D) NHEJ efficiency was measured between Ku80+/+ and Ku80−/− iPSC lines.
(E) Loss of Ku80 abolishes the stimulatory effect of Sirt6 on NHEJ. FLAG-control or FLAG-Sirt6 viruses were introduced into Ku80+/+ or Ku80−/− iPSC lines.
(F) NHEJ efficiency was measured in a Sirt6−/− iPSC line infected with FLAG-control, FLAG-Sirt6, or FLAG-sirt6 mutants (H133Y).
(G) NHEJ efficiency was measured in a Ku80−/− iPSC line infected with HA-control, HA-ku80 or HA-Ku80Δ10.
(H) Ku80Δ10 abolishes the stimulatory effect of Sirt6 on NHEJ. FLAG-control or FLAG-Sirt6 viruses were introduced into HA-Ku80 rescued Ku80−/− or HA-Ku80Δ10 rescued Ku80−/− iPSC lines.
The cell lines in (A)–(C) were analyzed at passages 7–10 and at least 50 cells were quantified for each cell line. Bar graphs represent mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ns, no significant differences according to Student’s t tests in (B)–(D) and one-way ANOVA with Tukey-Kramer post hoc test in (E)–(H) (n = 3 independent experiments). See also Figure S5.
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7. Figure 5
Ku80 Is Responsible for Sirt6-Induced Phosphorylation of DNA-PKcs on Ser2056
(A) Cells were infected with lentivirus FLAG-Sirt6 and FLAG-Control to establish Sirt6 overexpression and control iPSC lines. Ku80 coIP was performed in Sirt6+/+ and Sirt6−/− iPSC lines, and iPSC lines overexpressed control or Sirt6. Membranes were immunoblotted for Ku80 and Ku70.
(B) DNA-PKcs coIP was performed in Sirt6+/+ and Sirt6−/− iPSC lines, and iPSC lines overexpressed control or Sirt6. Membranes were immunoblotted for Ku80 and DNA-PKcs.
(C) Immunoblotting showing DNA-PKcs phosphorylation at Ser2056 in Sirt6+/+ and Sirt6−/− iPS lines after treatment with or without etoposide for 0.5 hr.
(D) The Sirt6-induced autophosphorylation of DNA-PKcs/S2056 was blocked by the DNA-PKcs inhibitor Nu7026.
(E) The Sirt6-induced autophosphorylation of DNA-PKcs/S2056 was attenuated by Ku80 deficiency.
(F) Immunoblotting showing DNA-PKcs phosphorylation at Ser2056 in Ku80+/+ and Ku80−/− iPSC lines after treatment with or without etoposide for 0.5 hr.
(G) Immunoblotting showing DNA-PKcs phosphorylation at Ser2056 in Ku80−/− iPSC clones infected with HA-Control, HA-Ku80, or HA- Ku80Δ10 after treatment with etoposide for 0.5 hr. Black arrows indicate specific bands.
Cell Reports  , DOI: ( /j.celrep )
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8. Figure 6
Sirt6 Improves the Stability of iPSCs Generated from the Skin Fibroblasts of Old Mice
(A) Representative images of AP staining and immunostaining for Oct4 and Nanog in OSKM+Sirt6 iPSCs derived from old mice. Nuclei were stained with Hoechst. Black scale bar, 100 μm; white scale bar, 10 μm.
(B) Representative alkaline comet assays of OSKM and OSKM+Sirt6 old mouse iPSC lines.
(C and D) Tail moment (C) and percentage of DNA content in tails (D) of six OSKM and six OSKM+Sirt6 old mouse iPSC lines. The 12 cell lines were analyzed at passages 7–10. For each cell line, at least 50 cells were quantified.
(E) NHEJ efficiency was measured in OSKM and OSKM+Sirt6 iPSC lines derived from old mice.
Bar graphs represent mean ± SEM. p Values are according to Student’s t tests. See also Figure S6.
Cell Reports  , DOI: ( /j.celrep )
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