**2. PPAR-γ ligand-binding activity**

PPAR-γ ligand-binding activity was assessed using a GAL-4-PPAR-γ chimera assay system (**Figure 1**) [3]. CV-1 monkey kidney cells from the American Type Culture Collection (ATCC) were suspended in Dulbecco's Modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 50 IU/mL Penicillin G sodium salt, 50 μg/mL streptomycin sulfate, and 37 mg/L ascorbic acid. The cells were then inoculated into a 96-well culture plate at 6 × 10<sup>3</sup> cells/well and incubated in 5% CO2 /air at 37°C for 24 h. Cells were washed with OPTI-minimum essential medium (MEM) and pM-hPPAR-γ and p4 × UASg-tk-luc were transfected into cells using LipofectAMINE PLUS (Gibco). pM and p4 × UASg-tk-luc were transfected into CV-1 cells as a mock control. Twenty-four hours after transfection, the medium was changed to DMEM containing 10% charcoal-treated FBS [4] and the cells were further cultured for 24 h. The cells were then washed with phosphate-buffered saline containing Ca2+ and Mg2+, and luciferase activity was measured using LucLite (Perkin-Elmer). Luminescence intensity was measured using a TopCount Microplate scintillation/luminescence counter. PPAR-γ ligand-binding activity was expressed as the relative luminescence intensity (test group/control group) determined for each sample.

**Figure 1.** GAL4-PPAR-γ chimera assay system.
