Evaluating PPAR Agonists and Antagonists on NHK

NHKs were seeded at 1.6 × 10⁵ cells/well in 6-well culture plates and were maintained in Humedia-KG2 at 37 °C in a 5% CO₂ atmosphere. After overnight incubation, the medium was exchanged with proliferation medium (Humedia-KG2, final Ca²⁺ concentration: 0.15 mM) or differentiation medium (excluding insulin, hEGF, hydrocortisone and BPE from Humedia-KG2 and final Ca²⁺ concentration: 1.5 mM), and then HC (final concentration of 200 µg/mL), agonists of PPARs (WY14643, GW501516 and troglitazone at final concentrations of 20, 20 and 5 µg/mL, respectively) or antagonists of PPARs (GW6471, GSK0660 and GW9662 at final concentrations of 4, 20 and 20 µg/mL, respectively) dissolved in 0.2% dimethyl sulfoxide were added to the medium. An amount of 0.2% dimethyl sulfoxide was added as a control. The NHKs were then cultured for 6, 12, 24 or 48 h and analyzed using real-time RT-PCR.