FIGURE 4.

PPARγ binds and activates the gene promoter of Fam57b variant 2. A, schematic illustration of Fam57b variant 2 promoter and luciferase reporter construct. The five candidates for the PPRE are numbered 1–5 in the promoter region of Fam57b variant 2. These promoter sequences were inserted into the pGL4.10 promoterless reporter vector. The sequences of WT and mutant types (mut1 and mut2) for the second PPRE are indicated. B, NIH3T3 cells were transfected with the reporter vectors together with PPARγ or the control expression vector and treated with rosiglitazone (Rosi) at 24 h post-transfection for another 24 h. The cells were harvested, and luciferase activity was measured with ARVO according to the manufacturer's instructions. The luciferase activity was shown as FL/RL. FL, firefly luciferase activity was normalized by RL, renilla luciferase. C, the pro1–5 wild type (pro_wt), pro1–5 mutants 1 and 2 (pro_mut1 and pro_mut2), and pro1–5 PPRE2 deleted (pro_del) were analyzed as described above. D, EMSA was performed using a ³²P-labeled oligonucleotide containing the above second PPRE WT and mut1 of Fam57b var2 promoter and ap2 PPRE as a positive control. The labeled probes were incubated with nuclear extracts of adipocytes (Ad NE) and preadipocytes (Pread NE) of ST2 cells as a negative control. For the supershift assay, the adipocyte nuclear extract was preincubated with labeled WT or ap2 PPRE and then incubated with an anti-PPARγ antibody or mouse IgG for 20 min. The DNA-protein complexes were resolved by PAGE. Lane 1, free probe; lanes 2–4, WT, mut1, and ap2 PPRE with preadipocyte nuclear extract, respectively; lanes 5–7, WT, mut1, and ap2 PPRE with adipocyte nuclear extract, respectively; lanes 8 and 10, WT and ap2 with mouse IgG and Ad NE, respectively; lanes 9 and 11, WT and ap2 PPRE with PPARγ-specific antibody and adipocyte nuclear extract, respectively. Results are means ± S.D. (error bars) (n = 3).