Fig. 5.

SIRT1 increases ATGL promoter activity and regulates binding of FoxO1 to the ATGL promoter. A: HEK 293T cells were transiently transfected with −2,979/+21 luciferase ATGL promoter construct together with enhanced green fluorescence protein. Cells were cotransfected with either empty vector, SIRT1 or FoxO1, or with both SIRT1 and FoxO1 cDNA. After 48 h, cells were washed three times in cold PBS and harvested in the reporter lysis buffer. Luciferase activity in cell lysates was assayed as described in MATERIALS AND METHODS and normalized by GFP fluorescence. Data are presented in triplicate as mean ± SD. * P < 0.05; NS, not significant. A representative result of three independent experiments is shown. B: Control (EV) and FoxO1-infected MEFs were homogenized, and total cell lysates (50 µg) were analyzed by Western blotting. Actin was used as loading control. C: Control (EV) and FoxO1-infected MEFs were transiently transfected with −2,979/+21 LUC ATGL promoter construct together with eGFP. Cells were cotransfected with either empty vector or SIRT1 cDNA. After 48 h, luciferase activity in cell lysates was assayed and normalized by GFP fluorescence. A representative result of two independent experiments is shown. Data are presented in triplicate as mean ± SD. * P < 0.05; NS, not significant. D, E: Chromatin immunoprecipitation assay was performed in control (EV) and SIRT1-ablated (Sh) 3T3-L1 adipocytes after 4 h serum withdrawal. Following cross-linking and sonication, genomic fragments were immunoprecipitated with antibody against FoxO1 or rabbit IgG and amplified by PCR (D) or by SYBR green reaction (E) as described in MATERIALS AND METHODS. Data are representative of three independent experiments and are expressed as mean ± SD relative to the promoter binding in EV cells. * P < 0.05.