Figure 4. ATP binding suppresses Sirt1 activity in vivo.

(a) Sirt1 interaction with substrate is regulated by ATP in vivo. FLAG-tagged p65 was co-expressed in 293 HEK cells with either V5-tagged HY mutant or 2A HY double mutant Sirt1. Co-immunoprecipitation was performed by immunoprecipitating with anti-V5 antibody followed by immunoblotting with either anti-FLAG antibody for detection of bound protein or anti-V5 antibody for immunoprecipitants. (b) Sirt1 KO MEFS stably expressing V5-tagged HY, WT or 2A Sirt1 were incubated with media containing either 25 mM Glu or 25 mM 2-DG for 2 h and acetylation of p65 was monitored by immunoblotting whole-cell extracts with acetyl p65 (K310) antibody. Four independent experiments were performed and data are represented as mean±s.e.m. (c) Sirt1 expressing MEFS from b were exposed to heat shock (42 °C, 30 min) and cell viability was determined after 24 h later by Trypan-blue exclusion assay (n=6). (d) Representative images of Oil Red O staining of cells stably expressing WT or 2A mutant Sirt1 4 and 6 days after addition of adipogenic cocktail (left). Adipocyte differentiation was quantified at 4 and 6 days by Oil Red O extraction (right) (n=3). (e) qRT-PCR analysis of adipogenic gene mRNA in differentiated WT and 2A mutant Sirt1 cells from (d). Three independent experiments were performed. *P<0.05; **P<0.01; ***P<0.001. two-tailed unpaired Student's t-test was used for statistical calculation.