Fig 1: Serum biochemistry suggests comparable recovery on normal diet in WT1 and KO1 after 2-week choline-deficient, ethionine-supplemented (CDE) diet and continued lack of ß-catenin in KO1.(A) Experimental design showing WT1 and KO1 on 2 weeks of CDE diet and recovery on normal diet for up to 6 months with analysis at intermediate time points as indicated. (B) Serum alanine aminotransferase (ALT), bilirubin, and alkaline phosphatase (ALP) in the two groups over time (one-way ANOVA, **p<0.01, ****p<0.0001, n = 3–5 per group). (C) ß-Catenin immunohistochemistry in WT1 and KO1 mice at 3 months and 6 months of recovery showing absence of ß-catenin in biliary epithelial cells (BECs) and hepatocytes in KO1 (100×). (D) Ctnnb1 gene expression in WT1 and KO1 mice during recovery from CDE diet shows continued ß-catenin absence over time in KO1 (one-way ANOVA, *p<0.05, **p<0.01, ****p<0.0001, n = 3–5 per group, individual animal values represented by dots).
Fig 2: Comparable recovery of WT2 and KO2 on normal diet after initial 2-week choline-deficient, ethionine-supplemented (CDE) diet injury, along with repopulation of KO2 livers with biliary epithelial cell (BEC)-derived ß-catenin-positive hepatocytes.(A) Experimental design showing WT2 and KO2 on 2 weeks of CDE diet and recovery on normal diet for up to 6 months with analysis at intermediate time points as indicated. (B) Serum alanine aminotransferase (ALT), bilirubin, and alkaline phosphatase (ALP) in the two groups over time (one-way ANOVA, **p<0.01, ****p<0.0001, n = 3–6 per group). (C) ß-Catenin immunohistochemistry in WT2 and KO2 mice at 3 months and 6 months of recovery showing ß-catenin-positive BECs and hepatocytes in KO2 and WT2 (100×). (D) Ctnnb1 gene expression in WT2 and KO2 mice during recovery from CDE diet (one-way ANOVA, *p<0.05, **p<0.01, ****p<0.0001. n = 3–6 per group; individual animal values represented by dots).
Fig 3: Modulation of ß-catenin in biliary epithelial cells (BECs) perturbs its complex with p65 to impact NF-?B activity.(A) Luciferase reporter assay shows successful knockdown of Ctnnb1 in small cholangiocyte cell (SMCC) line by TOPFlash assay (left), which stimulates p65 transcriptional activity with or without 100 ng/ml lipopolysaccharide (LPS) (right) (unpaired t-test, ns: no significance, *p<0.05, **p<0.01, ****p<0.0001, n = 3 biological replication). (B) Luciferase reporter assay shows expression of constitutively active S45Y-ß-catenin enhances TOPFlash (left) and suppresses p65 transcriptional activity with or without 100 ng/ml LPS (right) (unpaired t-test, ns: no significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n = 3 biological replication). (C) Representative WB from two independent experiment shows knockdown of Ctnnb1 increases p65 nuclear translocation with or without 500 ng/ml LPS. (D) Quantification of nuclear ß-catenin (left) and nuclear p65 (right) to HH3 (blots in (C) were technically quantified three times and p-value was calculated using unpaired t-test, ns: no significance, **p<0.01, ***p<0.001, ****p<0.0001). (E) Identification of RELA and NFKB1 among the top 15 transcription factors identified by applying the 335 differentially expressed genes (DEGs) to JASPAR. (F) qPCR shows knockdown of Ctnnb1 in SMCCs induces Ccl2 (left) and Cxcl5 (right) expression (unpaired t-test, ns: no significance, *p<0.05, **p<0.01, n = 3 biological replication). (G) qPCR shows Ccl2 (left) and Cxcl5 (right) are induced in KO1 after 6-month recovery of choline-deficient, ethionine-supplemented (CDE) diet (unpaired t-test, *p< 0.05, n = 3–4 biological replication). (H) Representative immunoprecipitation (IP) image from two independent experiment shows p65 is strongly associated with ß-catenin in SMCC. (I) IP shows that p65 is associated with ß-catenin in whole liver lysate (L: liver; S: SMCC; P: equal amount of liver and SMCC lysate). (J) Quantification of colocalization of p65 and ß-catenin is significantly diminished in KO1 compared to WT1 (unpaired t-test, **p<0.01, n = 3–4 biological replication). Figure 7—source data 1.WB shows knockdown of Ctnnb1 increases p65 nuclear translocation with or without 500 ng/ml lipopolysaccharide (LPS). Figure 7—source data 2.Immunoprecipitation (IP) image shows p65 is strongly associated with ß-catenin in small cholangiocyte cell (SMCC). Figure 7—source data 3.Immunoprecipitation (IP) shows that p65 is associated with ß-catenin in whole liver lysate.
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