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

Figure 5.

Figure 5.

(A) Schematic explaining the generation of muscle-specific SIRT6 knockout mice (mSIRT6-KO) using the ACTA-Cre and SIRT6 floxed mice. Western blotting analysis was performed to confirm the deletion of SIRT6 in heart and skeletal muscle of the knockout mice. SIRT6-Flox mice were used as controls. (B) Representative images of western blotting SUnSET analysis depicting changes in protein synthesis rates in skeletal muscle and heart of control and mSIRT6-KO mice. (C) Quantitative representation of puromycin incorporation in heart and skeletal muscle shown in Figure 5B. The results are expressed as the fold change relative to SIRT6 floxed controls. n = 6 mice per group. Data are presented as mean ± s.d, *P < 0.05. (D) Representative images of western blotting analysis of mTOR signalling in skeletal muscle lysates of control and mSIRT6-KO mice. n = 6 mice per group. (E) qPCR analysis of relative mRNA expression levels of mTOR signalling genes in skeletal muscle tissues of control and mSIRT6-KO mice. n = 4–7 mice per group. Data are presented as mean ± s.d, *P < 0.05. AKT1 was used as a positive control. HPRT was used as a negative control. (F) Heart weight to tibia length ratio (HW/TL) of age-matched control and mSIRT6-KO mice at 9 months of age. n = 8 mice per group, Data are presented as mean ± s.d, *P < 0.05. (G) Echocardiographic analysis of cardiac function in age-matched control and mSIRT6-KO mice at 9 months of age. Fractional shortening was measured as an indicator of cardiac function. n = 4 mice per group, Data are presented as mean ± s.d, *P < 0.05. (H) Representative immunofluorescence images showing ANP staining in control and SIRT6 depleted (SIRT6-KD) primary cardiomyocytes treated with vehicle or 100 nM Rapamycin for 24 h. ANP was stained red and was used as a marker of hypertrophy. Myomesin was stained green and was used as a marker for cardiomyocytes. The nuclei were stained blue with Hoechst 33342. Scale bar = 20μm. (I) The percentage of cells showing ANP staining in each group shown in Figure 5H was calculated and represented as a pie-chart. (J) Heart weight to tibia length ratio (HW/TL) measured in vehicle or Torin1 administrated control and mSIRT6-KO mice. n = 4–5 mice per group, Data are presented as mean ± s.d, *P < 0.05. (K) Fractional shortening measured before and after vehicle or Torin1 administration to control and mSIRT6-KO mice. n = 4–5 mice per group, Data are presented as mean ± s.d, *P < 0 05.