Pg5luc reporter

293T cells(ATCC, USA) were maintained in DMEM containing 10% fetal bovine serum (FBS) and were transiently transfected using Lipofectamine 2000 reagent (Invitrogen, USA). All mutant PPARγ plasmids were created using the Quick-Change site-directed mutagenesis kit (Stratagene, USA). Twenty-four-well plates were plated 24 hours prior to transfection (5 × 10⁴ cells per well). For the Gal4-driven reporter assays, the cells were transfected with 200 ng of Gal4-LBDs of various nuclear receptors and 200 ng of pG5Luc reporter (Promega, USA). Ligands were added five hours after transfection. Cells were harvested 24 h later for the luciferase assays. Luciferase activities were analyzed as the the instruction of CheckMate™ Mammalian Two-Hybrid System (Promega, USA).

The day before transfection, cells were trypsinized and transferred into wells of polystyrene 96-well culturing plates (Costar). Ten µL of a water solution containing 30 ng of the mutated Gal4-FusionRed-p65 construct, 0.1 µg of the pG5-Luc reporter (Promega), encoding the secreted Gaussia luciferase, and 0.2 µg pGl3-Basic vector was mixed with 1.5 µL of 1 mg/mL solution of PEI, pH = 7.0. The mixture was incubated for 10 min at room temperature for the efficient formation of DNA-PEI complexes and added directly to cells in plate wells containing 100 µL of the cultured medium. After transfection, cells were illuminated for 24 h with either blue pulsed light (array of 448 nm light-emitting diodes (LEDs), 30 s ON, 5 min OFF) or yellow-orange pulsed light (567 nm LED array, 30 s ON, 5 min OFF) at a light power density of 100 mW/cm².
For measurements of Gaussia luciferase activity, 1 µL of cell culture medium was mixed with 100 µL of 2 µM coelenterazine (Sigma, St. Louis, MO, USA) in wells of 96-well reflective bottom plates for bioluminescent assays (Stellar Scientific, Owings Mills, MD, USA). The quantification of bioluminescence was performed using a Victor X-5 plate reader (Perkin Elmer, Waltham, MA, USA). The contrast of 448 nm/567 nm reporter expression was calculated as luciferase activity in samples under illumination with 448 nm light divided by that at 567 nm light.