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. Author manuscript; available in PMC: 2015 Apr 17.
Published in final edited form as: Nat Med. 2012 Oct 21;18(11):1643–1650. doi: 10.1038/nm.2961

Figure 5.

Figure 5

SIRT6 is a co-repressor of c-Jun transcriptional activity. (a) ChIP analysis to detect H3K9 acetylation at the promoters of the indicated IGF signaling–related genes in WT and SIRT6 knockout heart samples. Antibodies to acetylated H3K9 (AcH3K9) and to H3 were used, and acetylated H3K9 levels are shown relative to total H3 levels. n = 4 independent experiments. Data are presented as the mean ± s.d. (b) SIRT6 binding to c-Jun, as determined by western blots of SIRT6 or control IgG antibody immunoprecipitates from rat cardiomyocytes. WCL, whole-cell lysate. (c) Luciferase activity in cell extracts from control or SIRT6 knockdown (SIRT6-KD) 293T cells transfected with a c-Jun–dependent luciferase reporter plasmid. n = 5 independent experiments. Data are presented as the mean ± s.d. RLU, relative light units. Student’s t test was applied to calculate the P value. *P < 0.001. (d) Luciferase activity in rat cardiomyocytes cotransfected with the c-Jun–dependent luciferase reporter plasmid and plasmids expressing WT SIRT6 (SIRT6-WT) or mutant SIRT6 (SIRT6-H133Y); the cells were serum starved overnight and induced with FBS or with the c-Jun activator phorbol 12-myristate 13-acetate (PMA). n = 8 independent experiments. Data are presented as the mean ± s.d. ANOVA was applied to calculate the P value. *P < 0.001. (e) ChIP analysis using a c-Jun–specific antibody to detect c-Jun occupancy at promoters of IGF signaling–related genes in WT or SIRT6 knockout mouse heart homogenates. n = 4 independent experiments. Data are presented as the mean ± s.d. *P < 0.001 compared to WT (Student’s t test). (f) ChIP analysis using a SIRT6-specific antibody to detect SIRT6 occupancy at the promoters of the indicated genes in control (WT) and c-Jun knockdown (c-Jun-KD) rat cardiomyocytes. SIRT6 occupancy at the promoters is shown relative to the background signal with IgG control antibody. *P < 0.001 (Student’s t test). n = 4 independent experiments. Data are presented as the mean ± s.d. (g) Western blots showing c-Jun knockdown in rat cardiomyocytes.