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Materials and methods
Chemical reagents. The citrus components used except auraptene were kindly provided by the National Institute of Fruit Tree Science, Ministry of Agriculture, Forestry and Fisheries (Shizuoka, Japan). Auraptene was purchased from LKT Lab (MN, USA). All the other chemicals used were from Sigma (MO, USA) or Nacalai Tesque (Kyoto, Japan).
Luciferase ligand assay. Luciferase ligand assay was performed using Cabozantinib advanced highly sensitive system, which was developed by modifying the dual luciferase system (Promega, WI, USA), as previously described [17] and [18]. Briefly, for assay using the GAL4/PPAR chimera system, we transfected p4xUASg-tk-luc (a reporter plasmid), pM-hPPARα, pM-hPPARγ or pM-hPPARδ (an expression plasmid for a chimera protein for a GAL4 DNA binding domain and each human PPAR ligand binding domain) and pRL-CMV (an internal control) into CV1 cells cultured on 24-well plates. The transfection was performed using LipofectAMINE (Invitrogen, Corp., Carlsbad, CA, USA) according to the manufacturer′s protocol. Twenty-four hours after the transfection, the transfected cells were cultured in a medium containing each compound for an additional 24 h. Luciferase ligand assay was performed using the dual luciferase system in accordance with the manufacturer′s protocol.