Fig. 6: HDCA inhibits nuclear export of PPARα in an FXR-independent manner.

a Luciferase assay of HDCA on FXR activity in HEK293T cells (n = 3). After 24 h co-transfection with pCMV-Script-hFXR and PGL4-Shp-TK firefly luciferase plasmids, followed by continuous 6 h starving in 1% FBS DMEM medium, the HEK293T cells were exposed to DMSO or HDCA (20 μM and 100 μM) for 24 h to test FXR activity. The left panel was cell viability test, and the right panel was Luciferase assay on FXR activity. Difference between groups were determined by one-way ANOVA test followed by Tukey’s multiple comparison. b mRNA expressions of FXR downstream and fatty acid oxidation related genes treated by FXR antagonist, Guggulsterone (20 μM) with or without HDCA (100 μM) for 24 h in AML12 cells (n = 3). Difference between groups were determined by unpaired two-tailed Student’s t test. c Immunofluorescence of PPARα with Guggulsterone (20 μM) and/or HDCA (100 μM) treatment for 24 h in AML12 cells (Scale bar, 50 μm) (Representative images are shown for each condition from one of three biologically independent experiments.). d Immunofluorescence of PPARα with Importazole (IPZ, 10 μM), Leptomycin B (LMB, 10 ng/μl), and/or HDCA (100 μM) treatment in AML12 cells. AML12 cells were pretreated with IPZ for 1 h and then treated with HDCA for 24 h or treated with LMB and HDCA combination for 24 h (Scale bar, 25 μm). Difference between groups were determined by unpaired two-tailed Student’s t test (Representative images are shown for each condition from one of three biologically independent experiments.). e Total, nuclear, and cytoplasmic protein levels of CRM1, RAN, and PPARα after HDCA treatment (100 μM) for 24 h in AML12 cells (n = 3). Difference between groups were determined by unpaired two-tailed Student’s t test. f Total, nuclear, and cytoplasmic protein levels of CRM1 and RAN in the liver of mice treated as in Fig. 2a (n = 6 per group). Difference between groups were determined by one-way ANOVA test followed by Tukey’s multiple comparison. The findings in a–b and e were confirmed in two independent experiments. Data are presented as mean values ± SEM. The average of gene and protein expression in control group is normalized as 100%. Source data are provided as a Source Data file.