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. 2009 Aug 18;284(42):28762–28774. doi: 10.1074/jbc.M109.034165

FIGURE 5.

FIGURE 5.

Involvement of SIRT1 in SMILE repression of ERRγ. Reporter assays in A–D, H, and I were performed as described under “Experimental Procedures.” The means ± S.D. (n = 3) of a representative experiment are shown. HepG2 cells were cotransfected with 0.1 μg of indicated reporter plasmids, (HNF4)8-Luc (A), or sft4-Luc (B–D) and 0.1 μg of pcDNA3-HA-HNF4α (A) or pcDNA3-FLAG-ERRγ (B–D), together with or without pcDNA3-FLAG-SMILE (0.2 μg in A–C, and 0.1 μg in D), and indicated doses of pcDNA3-Myc-SIRT1 or -SIRT1H363Y. 36 h after transfection, the cells in A–C were left untreated or treated with HDAC inhibitor TSA or SIRT1 inhibitors sirtinol (20 μm), EX527 (10 μm), or nicotinamide (NAM, 20 mm) for 12 h prior to the measurement of luciferase activity. E, in vitro interaction of SMILE and SIRT1. 35S-Radiolabeled SIRT1 protein was incubated with GST or GST-SMILE or GST-ERRγ fusion proteins. Protein interactions were detected via autoradiography. F, in vivo interaction of exogenous SIRT1 and SMILE. HepG2 cells were cotransfected with wild-type pcDNA3-myc-SIRT1 or pcDNA3-myc-SIRT1H363Y and pEBG-SMILE (GST-SMILE) or pEBG alone (GST). Protein interactions were examined via in vivo GST pulldown. The top and middle panels (GST puri) show GST bead-precipitated Myc-SIRT1 and GST fusions, respectively. The bottom panel shows the protein expression levels of Myc-SIRT1 in cell lysates. G, in vivo interaction of endogenous SIRT1 and SMILE. Coimmunoprecipitation assays were performed with cell extract from HepG2 cells using anti-SMILE antibody. Endogenous SMILE was immunoprecipitated with SIRT1 (upper panel). The proteins in the cell lysates (middle and lower panels) were analyzed by Western blot (WB) analysis using indicated antibodies. H and I, HepG2 cells were transfected with pSUPER (control (Con)) or pSUPER siSMILE-I (siSM#1), or pSUPER siSMILE-II (siSM#2), or pSUPER siSIRT1 (siSIRT1). After 24 h, the cells were cotransfected with expression vector for FLAG-ERRγ and sft4-Luc reporter vectors. 36 h after the second transfection, the cells were treated with or without indicated SIRT1 activators Resveratrol (100 nm) or piceatannol (20 μm) for 12 h prior to the measurement of luciferase activity. J, effect of siSMILE and siSIRT1 on the expression of SMILE and SIRT1. HepG2 cells were transfected with pSUPER siSMILE-I (siSM#1), siSMILE-II (siSM#2), siSHP or pSUPER (Con), and after 72 h the total RNA was isolated. The mRNA levels of SHP and SMILE were measured via RT-PCR analysis with β-actin shown as a control. The data shown are representative of at least three independent experiments.