Fig. 2

Transcriptional activity of recurrent PPARγ mutants. a A reporter plasmid containing the firefly luciferase gene under the control of a PPRE-X3-TK promoter was co-expressed in HEK293FT cells with a pcDNA3 vector encoding wild-type (WT) or mutant (P113S, R164W, R168K, S249L, M280I, I290M, T475M) PPARγ2. Renilla luciferase, expressed under the control of the CMV promoter, was used to normalize the signal. The data shown are the means ± SD of one representative experiment conducted in sixtuplate. The results for each mutant were compared with those for the WT in Dunnett’s multiple comparisons test, *0.01 < p < 0.05; ****p < 0.0001. b 5637 cells were transiently transfected with a pcDNA3 vector encoding WT or mutant (P113S, R164W, R168K, S249L, M280I, I290M, T475M) PPARγ. The expression of all PPARγ forms was checked by western blotting and quantified by RT-qPCR (Supplementary Fig. 1B). The effect of WT PPARγ2 expression on three PPARγ target genes was evaluated by RT-qPCR (Supplementary Fig. 1C). The expression of PPARγ target genes was normalized against PPARγ expression and is expressed as percentage of stimulation relative to the expression induced by WT PPARγ. The data are presented as the mean ± SD of four independent experiments. The results for each mutant were compared with those for the WT in Dunnett’s multiple comparisons test: *0.01 < p < 0.05; **0.001 < p < 0.01; ***0.0001 < p < 0.001; ****p < 0.0001. c PPARγ knockdown with three different siRNAs in RT4 cells harboring the PPARγ T475M mutation. PPARγ expression was evaluated by western blotting (lower left panel) 96 h after transfection, living cells were counted (upper left panel) 24, 48, 72, and 96 h after transfection. Data are presented as means ± SD of three independent experiments performed in duplicate. The results for each mutant were compared with those for the WT in Dunnett’s multiple comparisons test: *0.01 < p < 0.05; **0.001 < p < 0.01; ***0.0001 < p < 0.001; ****p < 0.0001. The expression levels of three PPARG target genes were assessed by RT-qPCR for two independent experiment at 48 and 96 h after transfection (right panel). Data for each experiment are represented. a–d Source data are provided as a Source Data file