SIRT1 expression correlates with c-Myc expression in Tet-O-MYC cells and influence on proliferation of cells.

<p>A) SIRT1 expression directly correlates with c-MYC in a time dependent manner. When oncogenic c-Myc expression is restored over five days (Re-ON), increased SIRT1 expression returns. B and C) Quantitative real time PCR (qPCR) and Western blot. After transfection of Tet-O-MYC cells with a SIRT1 over-expression plasmid, the expression of SIRT1 mRNA and protein as well as c-Myc significantly increases compared with control vector (CV). Knock down of SIRT1 by shRNA against SIRT1 resulted in decreased expression of mRNA and protein for SIRT1 and c-Myc, as well as increased acetylation of p53 protein (ac-p53) and increased expression of p21. D) SIRT1 overexpression promotes increased proliferation. Knock-down of SIRT1 decreased proliferation as demonstrated by MTT and colony forming assay. E) Nicotinamide (NAM), a SIRT1 inhibitor, significantly decreases the proliferation of both MYC-ON and MYC-OFF cells. The MTT assay was performed after seeding of 2×10³ cells for two to three days. Colony forming assay were performed in the presence of 10 mM NAM. A colony-forming assay was performed by culture of 2×10³ cells for 7 days in 24-well culture plates. A single asterik indicates <i>P</i><0.05, two asteriks indicate <i>P</i><0.001.