Fig 2: ATAC-Seq and Transcriptomic Analysis Revealed a Uniquely Accessible WNT Gene Program in Squamous PDCLs that Are Tolerant to GSK3B Inhibition(A) Western blot (WB) for either HNF4A or GATA6 in representative PDCLs of the classical (pancreatic) or squamous subtype. 20 µg of the same protein lysate was probed with stated antibodies on different blots. Actin panel is a representative loading control (HNF4A loading shown). (Above) Oncoplot showing somatic mutations in genes involved in chromatin regulation. Green = structural variant (SV); purple = single-nucleotide variant (SNV) or indel.(B) Venn diagram showing the number of common and unique annotated gene peaks in PDCLs grouped by response to GSK3ßi. GSK3ßi resistant = PacaDD137, TKCC-22, Mayo 5289; GSK3ßi tolerant = TKCC-26, TKCC-06, TKCC-15, TKCC-18; GSK3ßi sensitive = TKCC-09, TKCC-10, TKCC-2.1.(C) ATAC-seq density plots of accessible genes in 10 PDCLs representative of the classical (pancreatic) or squamous subtypes.(D) ATAC-seq genomic tracks for WNT7A. Highlighted regions show subtype-specific genomic peaks. PDCLs are grouped based on response to GSK3ß inhibitor.(E) Chart showing the genomic distribution of ATAC-seq peaks in squamous PDCLs that are sub-grouped based on response to GSK3ßi.(F) KEGG pathway enrichment analysis of enriched pathways accessible in GSK3ß-tolerant squamous PDCLs found at intronic and distal promoter sites.(G) WNT7A expression in squamous PDCLs treated with GSK3b (TDZD-8) for 144 h. For all graphs: **p < 0.01; ***p < 0.0001.See also Figures S6 and S7 and Table S7.

Fig 3: HNF4α transcriptionally regulated KLF4 and CDX2 in gastric epithelial cells.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.

Fig 4: CR2 KO embryos exhibit impaired pancreatic bud formation and failure to establish the hepato-pancreato-biliary system(A) RT-qPCR analysis of relative mRNA expression in CR2 deletion line embryos for hepatic, Hnf4; ductal, Sox9; and pancreatic, Pdx1 markers. WT, wild type; Het, heterozygous; ΔCR2, CR2-null embryos. E9.5, n ≥ 6; E11.5, n = 3. Data are normalized to Gapdh. Counts represent the mean ± SEM analyzed by a one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.(B) Representative light sheet microscopy images of E9.5 control (Sox17GFPCre/+;R26LSL.TdTomato/+ – center left) and CR2-deficient (Sox17GFPCre/ΔCR2;R26LSL.TdTomato/+ – center right) embryos. Reconstruction of posterior foregut surface visualizes hepatopancreatobiliary bud formation in the presence (hepatic bud, white; pancreatobiliary bud, green) and the absence of CR2 (lack of visible pancreatobiliary bud, arrow head). Scale bar, 400 μm.(C) Representative immunofluorescence staining of E9.5 WT and CR2-null embryonic sections from the dashed square region for FOXA2 (endoderm, green), HNF4A (liver bud, LB, magenta), and PDX1 (dorsal bud, DP/Proto-DP, and ventral pancreatic bud, VP, white). n = 3. Scale bar, 50 μm.

Fig 5: DTR-mediated specific ablation of Cyp2e1+peri-central hepatocytes.A, a cartoon figure showing the mating strategy for generation of Cyp2e1-DreER;R26-LR-tdT-DTR double transgenic mice. B, a schematic figure showing the genetic recombinations of the R26-LR-tdT-DTR allele with Cyp2e1-DreER and AAV8-TBG-Cre. C, a schematic figure showing experimental design. D, whole-mount epifluorescence view of livers collected from mice treated with DT or PBS. Yellow scale bar represents 2000 μm. E and F, immunostaining for tdTomato, HNF4a (E), or DTR (F) on liver sections treated with PBS. White scale bar represents 100 μm. C, central vein. G, immunostaining for CYP2E1, tdTomato, and E-cad on liver sections from mice treated with PBS or DT. White scale bar represents 100 μm. H, quantification of the percentage of tdTomato+ hepatocytes (DT group) in comparison of tdTomato+ hepatocytes (PBS group). I, immunostaining for Tunel and tdTomato on liver sections from mice treated with PBS or DT. White scale bar represents 100 μm. J, immunostaining for E-cad and tdTomato on kidney sections of Cyp2e1-DreER;Rosa26-rox-stop-rox-tdTomato (R26-R-tdT) mice and Cyp2e1-DreER;R26-LR-tdT-DTR. Both the mice received tamoxifen. White scale bar represents 100 μm. K, a cartoon figure showing Cyp2e1-DreER targeted hepatocytes specifically by using R26-LR-tdT-DTR mice and AAV8-TBG-Cre injection, while Cyp2e1-DreER targeted hepatocytes and kidney epithelial cells by using R26-R-tdT. Each image is representative of five individual biological samples. DTR, diphtheria toxin receptor; DT, diphtheria toxin.