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. 2019 Jan 22;54(3):879–892. doi: 10.3892/ijo.2019.4692

Figure 2.

Figure 2

FXR is involved in the regulation of bile acid-induced CDX2 and MUC2 expression. (A) Dual-luciferase reporter assay was conducted to determine the effect of knockdown of CDX2 expression on bile acid-enhanced MUC2 promoter activity. (B) Western blotting was conducted to determine the effect of knockdown of CDX2 on bile acid-enhanced MUC2 expression. (C) Comparison of CDX2 and MUC2 protein levels in each group of GES-1 cells. (D) Mutagenesis of the MUC2 promoter was performed using a site-directed mutagenesis kit. A dual-luciferase reporter assay was conducted to detect the luciferase activities in GES-1 cells transfected with a MUC2 promoter reporter construct or mutated construct. (E) A quantitative chromatin immunopre-cipitation assay was performed to investigate the effect of bile acids on the binding of CDX2 to the MUC2 promoter. (F) Reverse transcription-quantitative polymerase chain reaction was conducted to detect the mRNA expression levels of FXR in cells treated with DMSO, or CDCA or DCA at various concentrations (0, 100, 150 and 200 µmol/l) for 24 h. (G) Western blot analysis of FXR protein expression in each group of bile acid-treated GES-1 cells. (H) Comparison of FXR protein levels in each group of GES-1 cells stimulated with CDCA or DCA. (I) siRNA-FXR was transfected into cells with the CDX2-luc promoter construct. A dual-luciferase reporter assay was conducted to determine the effect of the downregulation of FXR on bile acid-enhanced CDX2 promoter activity. (J) Effects of treatment with the FXR agonist GW4064 on bile acid-enhanced CDX2 promoter activity. (K) Western blotting was performed to investigate the effect of FXR downregulation on bile acid-induced CDX2 and MUC2 expression. (L) Comparison of CDX2, MUC2 and FXR protein expression in each group of GES-1 cells. 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; FXR, farnesoid X receptor; MT, mutated type; MUC2, mucin 2; NC, negative control; n.s., not significant; sh, short hairpin RNA; si/siRNA, small interfering RNA; WT, wild-type.