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. 2011 Aug 12;12(10):1069–1076. doi: 10.1038/embor.2011.151

Figure 2.

Figure 2

SIRT1 expression is transcriptionally activated by CREB. (A) SIRT1 mRNA in HepG2 cells stimulated for 4 h with 10 μM Fsk (n=4). pCREB and CREB protein levels were analysed by western blot analysis. (B) HepG2 cells were transfected with a SIRT1 promoter luciferase reporter, a β-galactosidase normalization plasmid and 10 ng of pCMV–CREB or pCMV–KCREB. Luciferase activity was measured 48 h after transfection and values were normalized to β-galactosidase levels (n=4). Values are presented as fold increase over the empty vector (CMV; n=4). SIRT1 mRNA levels in (C) HepG2 cells transfected with pCMV–CREB or an empty vector (CMV; n=4) and (D) HepG2 infected with adenoviruses encoding GFP or the dominant-negative ACREB (n=6), after 4 h stimulation with 10 μM Fsk. Data are relative to control group. HepG2 cells were transfected with either (E) 10 ng of pCMV–CREB and serial deletions of the SIRT1 promoter luciferase reporter or (F) the SIRT1 promoter luciferase reporter containing site-directed mutagenesis of the indicated putative CREB-binding site. Luciferase activity was measured 48 h after transfection. (G) HepG2 cells were treated for 4 h with Fsk, and ChIP was performed using an IgG control or a CREB-specific antibody. Recruitment of CREB to the SIRT1 promoter was measured by qPCR using primers against the proximal −0.2 kb and the distal −3 kb promoter, and corrected for background measured in IgG immunoprecipitates (n=4). (H) SIRT1 mRNA abundance in liver from fed or 18 h-fasted mice infected with adenovirus expressing GFP or ACREB. Values are presented as the average±s.e.m., asterisk indicates statistical difference compared with control (A,C,D), WT SIRT1 promoter luciferase reporter (F) or fed group (G) and hash symbol indicates statistical difference compared with empty vector (B,E), distal promoter (G) or GFP (H) at P<0.05. ChIP, chromatin immunoprecipitation; CMV, cytomegalovirus; CREB, cyclic AMP response-element-binding protein; Fsk, forskolin; GFP, green fluorescent protein; IgG, immunoglobulin G; pCMV, CMV promoter; pCREB, CREB phosphorylation; prom, promoter; qPCR, quantitative PCR; RLU, relative luciferase units; WT, wild type.