Figure 4.

TR-FRET NRs binding or coactivator assays with 1a and related A₃ AR ligands. (A) TR-FRET competitive binding assays with 1a and 1b at 4 µM as ligands of PPARα, PPARγ, PPARδ, and GR were performed. In TR-FRET LXRα and LXRβ coactivator assays, 1a and 1b at 4 µM were evaluated to determine whether transactivation occurred. The positive controls were 19 for PPARα, 21 for PPARγ, 22 for PPARδ, dexamethasone for GR, and 20 for LXRα and LXRβ. DMSO in buffer was used as a blank control. (B) The kinase activity was evaluated by measuring the γ-³²P-ATP incorporation to CDK complexes. The inhibitory effects of 1a and 1c on the phosphorylation of CDK5/p25 and CDK5/p35 were tested at each K_m ATP concentration. DMSO was included in each negative control. Values were expressed in terms of percentage compared to each positive control. The TR-FRET-based competitive binding activities of troglitazone, 1a, 1b, 1c, and 3a to PPARα (C), PPARγ (D), and PPARδ (E) were evaluated. Pearson’s correlation coefficients (r²) between the binding affinities to PPARγ at 20 µM (F) or 4 µM (G) and relative adiponectin levels in the cell culture supernatants were calculated with RStudio software. Correlation coefficients between the binding affinities to PPARδ at 2 µM (H) or 0.4 µM (I) and relative adiponectin levels were calculated in the same way. Results are the mean ± SD of three independent experiments: (*) p ≤ 0.05 and (**) p ≤ 0.01.