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. 2019 Aug 2;47(17):9115–9131. doi: 10.1093/nar/gkz648

Figure 3.

Figure 3.

(A) qPCR analysis of relative mRNA expression levels of mTOR signalling genes in WT and SIRT6-KO mice heart tissues. n = 4–10 mice per group. Data are presented as mean ± s.d, *P < 0.05. AKT1 was used as a positive control. GAPDH was used as a negative control. (B) ChIP analysis to detect SIRT6 binding to the promoters of the indicated genes performed with a SIRT6-specific antibody or IgG control antibody in WT 293T cells. n = 3–4. Data are presented as mean ± s.d, *P < 0.05. (C) ChIP analysis to detect Sp1 binding to the promoters of the indicated genes performed with a Sp1-specific antibody or IgG control antibody in WT 293T cells. n = 3–4. Data are presented as mean ± s.d, *P < 0.05. (D) Representative images of western blotting analysis of mTOR signalling under Sp1 depleted (Sp1-KD) conditions in 293T cells. The Sp1 levels were depleted by transfecting the cells with specific shRNA and the knockdown was confirmed by western blotting. (E) Representative images of western blotting analysis of mTOR signalling in Sp1-KO HeLa cells. Sp1 was deleted using a specific guide RNA and the CRISPR-Cas9 system. (F) Representative images of western blotting analysis of mTOR signalling under Sp1 overexpression conditions in 293T cells. The cells were transfected with empty vector or Sp1 expressing plasmid for 48 h and Sp1 overexpression was confirmed by immunoblotting. (G) Sp1 was immunoprecipitated from 293T cells and the interaction with SIRT6 was tested by western blotting. IgG was used as control. Whole cell lysate (WCL) was probed for indicated proteins by western blotting. The results presented here are representative of three independent experiments. (H) Luciferase reporter assay to assess the transcriptional activity of Sp1 was performed in pcDNA, SIRT6-WT or SIRT6 H133Y expressing 293T cells. The results are expressed as the fold change relative to pcDNA expressing cells. n = 6. Data are presented as mean ± s.d, *P < 0.05. (I) Realtime qPCR analysis of relative mRNA levels of GLUT1 and GLUT3 in WT and SIRT6-KO mice heart tissues. n = 6 mice per group. Data are presented as mean ± s.d, *P < 0.05. (J) Sp1 was immunoprecipitated from the 293T cells transfected with pcDNA, SIRT6-WT or SIRT6-H133Y plasmids and western blotting analysis was performed to detect the levels of Sp1 acetylation (Ac-Lys) by anti-acetyl-lysine antibody. IgG was used as a negative control in this assay. Whole cell lysate (WCL) was probed for indicated proteins by western blotting. (K) Representative immunofluorescence images depicting the levels and localization of Sp1 transcription factor (green) under control and SIRT6-KD conditions in Hela cells. SIRT6 was depleted using specific siRNA and knockdown was confirmed by immunostaining for SIRT6 (green) in a parallel experiment (Supplemental Figure S2E). The nuclei were stained with Hoechst 33342 and are shown in blue. Scale bar = 20 μm.