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. 2020 Jul 6;6:56. doi: 10.1038/s41420-020-0290-3

Fig. 6. HNF4α transcriptionally regulated KLF4 and CDX2 in gastric epithelial cells.

Fig. 6

a AGS cells were transfected with shRNA lentiviral target HNF4α. Next, P1-HNF4α, P2-HNF4α, CDX2, KLF4, Villin1, and MUC13 were analyzed by western blotting (WB) and qRT-PCR. Error bar indicates the SEM, **P < 0.01 vs. negative control (NC), n = 3. b GES-1 cells were transfected with HNF4α2 overexpression lentiviral. HNF4α, KLF4, CDX2, MUC13, and Villin1 expression were analyzed by qRT-PCR and WB. Error bar indicates the SEM, **P < 0.01 vs. NC, n = 3. c GES-1 cells were transfected with HNF4α8 overexpression lentiviral. HNF4α, KLF4, CDX2, MUC13, Villin1, and ALPI mRNA was analyzed by qRT-PCR. Error bar indicates the SEM, **P < 0.01 vs. NC, n = 3. ALPI protein level was analyzed by WB. d KLF4 promoter fragment (2000 bp) from Ensemble was predicated using JASPAR tool. Reporter constructs containing the predicted HNF4-binding site and mutational site are shown (upper). Negative control (NC) and HNF4α2 overexpression plasmid was transiently transfected with these KLF4 promoter reporter constructs for 24 h and luciferase activity was assayed thereafter (lower). KLF4 promoter activity was expressed as fold induction (means ± SEM) compared with that of NC, n = 3. **P < 0.01. e GES-1 cells were treated with DCA (200 μM) for 24 h. Then, ChIP assay was performed to demonstrate the direct binding of HNF4α to the KLF4 promoter. DCA, Deoxycholic acid; M, Marker. f shHNF4α stably transfected AGS cells were transiently transfected with CDX2 promoter reporter constructs containing the predicted HNF4-binding sites for 24 h and luciferase activity was assayed. CDX2 promoter activity was expressed as the fold induction (means ± SEM) compared with that of NC. **P < 0.01, n = 3.