Figure 7
Binding of magnolol dimer (1), magnolol, and pioglitazone to the PPARγ ligand binding domain in a competitive in vitro assay (A). The different compounds were serially diluted in DMSO and incubated with purified human PPARγ LBD tagged with GST, a terbium-labeled anti-GST antibody, and fluorescently labeled PPARγ agonist (Fluormone™ Pan-PPAR Green). Compounds that bind to the PPARγ LBD displace the fluorescently labeled ligand leading to a fluorescence resonance energy transfer (FRET). Binding is estimated from the decrease of the emission ratio 520 nm/495 nm upon excitation at 340 nm. Data points are represented as mean ± SEM from four independent experiments performed in duplicate. K i values were calculated using a sigmoidal curve fit with variable slope and 95% confidence intervals are shown. Nuclear receptor-dependent luciferase reporter transactivation of PPARγ (B) and RXRα (C) by magnolol dimer (1) in comparison to magnolol. (B) PPARγ-mediated luciferase reporter gene transactivation by magnolol dimer (1), magnolol and pioglitazone. HEK-293 cells were transfected as described in the Methods section and stimulated for 18 h, as indicated. Luciferase activity was normalized to EGFP-derived fluorescence. Results are expressed as fold induction compared with the solvent control (DMSO, 0.1%). EC50 values were calculated using a sigmoidal curve fit with variable slope. (C) RXRα-mediated luciferase reporter gene transactivation by magnolol and the full RXR agonist 9-cis retinoic acid. HEK293 cells were transfected as described in the Methods section, treated as indicated for 18 h and luciferase activity measured as under (A). All data are shown as means ± SD of at least three independent experiments performed in quadruplicate. EC50 values were calculated and values are indicated together with 95% confidence intervals.
