XN down-regulates the TNF-induced NF-κB activation through FXR-independent mechanism. (A) Expression of the FXR receptor in various cell types. Whole-cell extracts were prepared and analyzed by Western blotting with an anti-FXR antibody. (B) XN and guggulsterone (GS) inhibit TNF-induced NF-κB expression in both MCF-7 and KBM-5 cells. Cells were pretreated with either 50 μM XN for 4 hours or 50 μM GS for 4 hours and then incubated with 0.1 nM TNF. Nuclear extracts were prepared and EMSA was performed. (C) Effect of XN on TNF-induced NF-κB expression in FXR-negative H1299 cells. Cells were pretreated with 50 μM XN for 4 hours and then incubated with 0.1 nM TNF. Nuclear extracts were prepared and EMSA was performed. (D) XN and GS inhibit bile acid induced NF-κB expression in MCF-7 cells. Cells were pretreated with either 50 μM XN for 4 hours or 50 μM GS for 4 hours and then incubated with 250 μM bile acids for 3 hours. Nuclear extracts were prepared and EMSA was performed. (E) The chemical structure of XN, an FXR ligand. A schematic representation of XN′s interaction with thiol group is also shown.