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. 2010 Aug 17;29(19):3395–3407. doi: 10.1038/emboj.2010.197

Figure 2.

Figure 2

Characterization of indole acetate-containing ligands as AF-2-mediated agonists for PPARγ. (A) Effects of each indole acetate-containing ligand on the activity of the Gal4DBD-PPARγLBD. The final concentrations of BRL49653 (BRL) and IDM were 1 and 10 μM, respectively. The concentrations of 5-HT, HIA, and MIA were 100 μM. The RLU (Luc/YFP) is shown with +s.d. (n=4). (B) Effects on the ligand-induced expression of the endogenous PPARγ-target genes AOX and LXRα in PMA-activated macrophage-like THP-1 cells. After activation by PMA, the cells were treated with 1 μM BLR49653 (BRL), 10 μM IDM, 100 μM HIA, and 100 μM MIA, respectively. β-ACTIN and GAPDH were used as references. (C) Effects on 3T3-L1 adipogenesis. After a pre-treatment with 1 μM Dex, 0.5 mM IBMX, and 5 μg/ml insulin, the cells were incubated with 10 μM 15d-PGJ2, 10 μM IDM, or 100 μM MIA, respectively. Oil red O stained cells are shown at the top and are quantified at the bottom by measurement at the OD550 with ±s.d. The statistical comparison of each ligand with a negative control (DMSO) was accomplished using the Mann–Whitney U-test. *P<0.05. n=4. (D) Effect of the Y473F mutation on the protein stability of Gal4DBD-PPARγLBD. The wild type (WT) and the Y473F mutant were transfected into HEK293T cells. After 1 day, the cells were collected and analysed by immunoblotting with an anti-Gal4 DBD antibody. Mock-transfected cells (-) were used as a negative control, and tubulin was used as a reference. (E) Effects of the Y473F mutation on the ligand-induced activity of Gal4DBD-PPARγLBD. Open and closed bars show the ligand-induced activities of the wild type (WT) and the mutant (Y473F), respectively. BRL49653 (BRL), IDM, and MIA were added at final concentrations of 1, 10, and 100 μM, respectively. The RLU (Luc/YFP) is shown with +s.d. (n=4). (F) Effects of the Y473F mutation on the IDM binding to the PPARγ LBD. The direct interactions of IDM with the wild type (WT) and the Y473F mutant PPARγ LBD were quantitatively analysed by ITC, as shown in the left and right panels, respectively.