Plasmid constructions and dual luciferase reporter assays.

For studies on the transcriptional activity of FGF21 and KLB genes, HepG2 hepatic cells and HIB1B adipogenic cells (48), respectively, were grown in 24-well plates and transiently transfected at ∼50% confluence with the corresponding promoter-luciferase reporter plasmids using Lipofectamine (Invitrogen). The reporter plasmid −1497-FGF21-Luc, containing a DNA fragment corresponding to positions −1,497 to +5 of the 5′ region of the mouse Fgf21 gene linked to the firefly luciferase gene, and a −77-FGF21-Luc mutant construct containing only the −77/+5 region of the FGF21 gene, were reported previously (48). The reporter plasmid driven by the KLB gene promoter, containing a DNA fragment corresponding to positions −1,055 to +45, was obtained from SwitchGear Genomics. Cells were also cotransfected with a pRL-CMV expression vector for Renilla luciferase (Promega). Each transfection condition was assayed in triplicates. The cells were incubated for 48 h after transfection and then incubated with or without drugs, as indicated in the text, for an additional 24 h before harvesting. Luciferase activity was measured on a Glomax 96 microplate luminometer using a dual luciferase reporter assay system kit (Promega). Promoter construct-driven luciferase activity was normalized to that of Renilla luciferase, used as a control for variations in transfection efficiency.