Fig. 6. Effect of LEF treatment on liver nuclear receptors. (A–D) The gene expression of nuclear receptors (Car, Fxr, Nrf2, Pxr, and PPARα) (A), downstream target gene expression (CYP2b10, Cyp7a1, HO-1, CYP3a11, CYP4a10, and CYP 4a14) (B), protein expression of Cyp4a10 and Cyp4a14 (proteomics data) (C), and the PPARα reporter luciferase assay (D). (A and B) Male C57BL/6 mice were administered LEF (40 mg/kg) or vehicle (PEG400) for 3 consecutive days, and then the mice were euthanized. Liver samples were collected and processed as previously described in the Materials and Methods. Gene expression was normalized against the endogenous control GAPDH. The data are representative of two independent experiments, and each point indicates the mean ± S.D. (n = 5). (C) Proteomics data showing the relative fold change from the control group (PEG400). Each point indicates the mean ± S.D (n = 5). (D) Cos-7 cells were transiently cotransfected with 200 ng plasmid containing the luciferase gene and 100 ng GAL4-PPARα LBD expression plasmid, and then the cells were treated with GFT-505 (a positive control) and LEF at the indicated concentrations for 24 hours. Luciferase activity was determined and normalized to Renilla luciferase activity. Each condition was performed with n = 3 for each experiment. As a control, the activity was measured in the presence of vehicle (DMSO). The data are representative of two independent experiments. Each point indicates the mean ± S.D. The raw data were log-transformed and the significant difference analysis was done on the transformed data. Differences between two groups were analyzed using the t test (*P < 0.05; **P < 0.01; ***P < 0.001). DMSO, dimethylsulfoxide; HO, heme oxygenase; Pxr, pregnane X receptor.