Figure 9

SIRT6 down-regulated Mstn expression via binding to NF-κB sites. (A) Schematic representation of mouse Mstn promoter depicting presence of two NF-κB binding sites and the primers (F and R) used for ChIP-qPCR²³. (B) SIRT6 occupancy at Mstn promoter was determined in proliferating C2C12 cells by ChIP assay. The PCR analysis was done for NF-κB sites in Mstn promoter. IgG-ChIP was used as negative control. (C) Bar graphs showing relative occupancy for SIRT6 at Mstn promoter as determined by ChIP analysis in C2C12 cells treated with vehicle, TNF-α or TNF-α+Bay11, a NF-κB inhibitor. Data are representative of three independent experiments and shown as mean ± SEM, *p < 0.0001. (D) In C2C12 cells transfected with mouse Mstn promoter-Luciferase reporter construct, luciferase reporter assay demonstrates that SIRT6 over-expression inhibits TNF-α-induced myostatin activity by suppressing NF-κB. Data are representative of experiments done in triplicates and shown as mean ± SEM, *p < 0.01.