Figure 3.

NF-κB is involved in the regulation of bile acid-induced FXR/CDX2/MUC2 signalling pathway activation. (A) Dual-luciferase reporter assay was conducted to determine the effect of bile acids on NF-κB activity. (B) Western blotting was conducted to detect the effects of bile acids on p50/p65 protein expression. (C) Comparison of p50/p65 protein levels in each group of GES-1 cells. (D) Dual-luciferase reporter assay was performed to detect the effect of the FXR agonist GW4064 or the antagonist Z-gug on bile acid-enhanced NF-κB activity. (E) Western blotting was conducted to determine the effect of the FXR agonist GW4064 or the antagonist Z-gug on bile acid-induced alterations in p50 and p65 protein levels. (F) Comparison of p50 and p65 protein levels in each group of GES-1 cells. (G) Dual-luciferase reporter assay was conducted to determine the effect of the NF-κB inhibitor PDTC on bile acid-enhanced CDX2 promoter activity. (H) Western blotting was performed to determine the effect of the NF-κB inhibitor PDTC on bile acid-induced alterations in CDX2 and MUC2 protein expression levels. (I) Comparison of CDX2 and MUC2 protein levels in each group of GES-1 cells. (J) Dual-luciferase reporter assay was conducted to detect the luciferase activities in GES-1 cells transfected with CDX2 promoter reporter construct or mutated construct. (K and L) Quantitative chromatin immunoprecipitation assay was performed to investigate the effect of the FXR agonist GW4064 or the antagonist Z-gug on the binding of NF-κB (K) p50 or (L) p65 protein to the CDX2 promoter. Data are presented as the means ± standard deviation from three independent experiments. ^*P<0.05, ^**P<0.01. CDCA, chenodeoxycholic acid; CDX2, caudal-related homeobox transcription factor 2; DCA, deoxycholic acid; DMSO, dimethyl sulfoxide; MT, mutated type; MUC2, mucin 2; NF-κB, nuclear factor-κB; n.s., not significant; PDTC, pyrrolidine dithiocarbamate; WT, wild-type; Z-gug, Z-guggulsterone.