Fig. 5.

FXR up-regulated TonEBP expression and nuclear translocation in primary cultured mouse IMCD cells. Primary MCD cells were treated with GW4064 (2.5 μM) and CDCA (50 μM) for 6 h or infected with Ad-FXRα2. (A) Real-time PCR analysis demonstrating mRNA expression of TonEBP in primary MCD cells isolated from WT mice. **P < 0.01, ***P < 0.001 vs. DMSO (n = 4). (B) Western blot assay showing that treatment of MCDs with GW4064 and CDCA markedly induced protein expression of FXR, TonEBP, and osmoprotective genes HSP70 and AR. (C) Quantitative PCR analysis showing that overexpression of FXR significantly up-regulated TonEBP mRNA levels in MCDs. *P < 0.05 vs. Ad-VP16 (n = 8). (D) Immunoblot assay demonstrating overexpression of FXR markedly induced FXR, TonEBP, HSP70, and AR expression. (E) FXR activation promoted nuclear translocation of TonEBP. Primary MCDs were treated with GW4064 and CDCA for 6 h. Confocal immunofluorescence was performed to detect TonEBP (red) nuclear translocation. The nucleus was visualized by staining with DAPI (blue). (Scale bar, 5 uM.) (F) FXR overexpression with Ad-FXRα2 markedly facilitated nuclear translocation of TonEBP. Cells infected with a control virus (Ad-VP16) had little effect on TonEBP nuclear localization. (Scale bar, 5 uM.)