FIGURE 2.

Effect of overexpression and knockdown of SMILE on ERRγ transactivation. Reporter assays (A–D) were performed as described under “Experimental Procedures.” A and B, effect of SMILE on ERRγ-mediated transcriptional activity. 293T and HepG2 cells were cotransfected with 0.3 μg of pcDNA3-FLAG-ERRγ, and 0.1 μg of sft4-Luc reporter vectors, together with indicated amounts of plasmids expressing WT SMILE, SMILE-L (SMILE-83Leu), and SMILE-S (SMILE-1Phe). C and D, siSMILE increases ERRγ transactivation. 293T and HepG2 cells were transfected with pSUPER (control (con)), or pSUPER siSMILE-I (siSM#1), or pSUPER siSMILE-II (siSM#2), or pSUPER siSHP (siSHP). After 24 h, the cells were cotransfected with expression vector for FLAG-ERRγ and sft4-Luc reporter vectors. The luciferase activity was measured 48 h after the second transfection. The means ± S.D. (n = 3) of a representative experiment are shown. **, p < 0.01, using Student's t test. E, effects of overexpressed SMILE on the protein levels of FLAG-ERRγ. 293T cells were cotransfected with various plasmids as indicated. The proteins of FLAG-ERRγ, SMILE, and tubulin were detected by respective antibodies though Western blot analysis. F, effect of siRNAs for SMILE or SHP on the expression of SMILE and SHP. 293T and 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.