Volume 143, Issue 6, Pages 1650-1659 (December 2012) Conditional Disruption of Axin1 Leads to Development of Liver Tumors in Mice  Gui Jie Feng, Welwyn Cotta, Xiao Qing Wei, Oliver Poetz, Rebecca Evans, Thierry Jardé, Karen Reed, Valerie Meniel, Geraint T. Williams, Alan R. Clarke, Trevor C. Dale  Gastroenterology  Volume 143, Issue 6, Pages 1650-1659 (December 2012) DOI: 10.1053/j.gastro.2012.08.047 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Generation of Axin1fl/fl mice. (A) Schematic of the Axin1 targeted allele. The AUG start codon and RGS domain of Axin1 are contained within exon 2. (B) Southern blot of targeted allele (gt) in ES cells showing the 10-kilobase band generated by a BamHI site that was introduced upstream of exon2. The Neo cassette was removed by FRT-mediated recombination. (C and D) PCR detection of alleles in mouse ear DNA. (E) Detection of Axin1 in the liver by Western blotting (R&D Systems, Abingdon, UK) and (F) immunoprecipitation (R&D Systems)/immunoblotting (Cell Signaling, Hertfordshire, UK). Gastroenterology 2012 143, 1650-1659DOI: (10.1053/j.gastro.2012.08.047) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Liver responses to the loss of Axin1. (A) Liver sections from mice 4 days after treatment with BNF showing H&E staining, immunostaining for the Ki-67 proliferation marker, and bromodeoxyuridine incorporation (20×; arrowheads indicate nuclear positivity). (B) Cohorts of mice (n = 4) were treated for 4 days with BNF and killed after 4 days or 3 months. Three to 5 fields per animal were counted for cell/field, nuclei diameter, and CV (central vein)/field. P values were determined using the Mann–Whitney U test. Gastroenterology 2012 143, 1650-1659DOI: (10.1053/j.gastro.2012.08.047) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Acute responses to the loss of Axin1. Liver sections were from mice 4 days after injection of BNF. (A) β-catenin and GS staining. ps, portal space; cv, central vein. Apcfl/fl/Cre mice are shown for comparison. (B) Liver extracts from cohorts of Axin1fl/fl/Cre (n = 11), Axin1wt/Cre (n = 15), and Apcfl/fl/Cre (n = 7) mice were analyzed for different β-catenin pools in technical triplicates. Signal intensities are displayed as a percentage (mean ± SD). Filled, open, and hatched bars correspond to WT, Axin1, or Apc mice, respectively. Gastroenterology 2012 143, 1650-1659DOI: (10.1053/j.gastro.2012.08.047) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Changes in gene expression following deletion of Axin1. Livers from cohorts of (A) BNF-treated Axin1fl/fl/Cre and Axin1wt/Cre mice (n = 4, 4 days) and (B) (n = 5, 3 months) after treatment with BNF were analyzed by quantitative RT-PCR for changes in gene expression. Data are expressed as fold change (Axinfl/fl/Cre:Axin1wt/Cre) (mean ± SD). (C) Cell proliferation rates 4 days and 3 months after deletion of Axin1 as determined by bromodeoxyuridine incorporation. Gastroenterology 2012 143, 1650-1659DOI: (10.1053/j.gastro.2012.08.047) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Prolonged effects of Axin1 gene deletion in liver. Axin1fl/fl/Cre and Axin1wt/Cre mice were injected with BNF (80 mg/kg) for 4 days and left for 3 months. Liver sections showing (A) H&E staining (20×) and immunohistochemistry for Ki-67 (20×) and (B) β-catenin (20×) and GS (10×) as indicated. Gastroenterology 2012 143, 1650-1659DOI: (10.1053/j.gastro.2012.08.047) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 HCC in Axin1fl/fl/Cre mice. Axin1wt/Cre and Axin1fl/fl/Cre mice (n = 9) were treated with BNF for 4 days and left for 1 year. Sections were stained with H&E (A–C, 20×) and for β-catenin expression (D and E, 20×; F, 40×). Axin1wt/Cre mouse livers show (A) a normal liver architecture and (D) absent nuclear β-catenin expression. Livers from Axin1fl/fl/Cre mice contain differentiated HCCs (B and C, E and F) whose expanding borders compress the adjacent parenchyma (arrows, C and E). Neoplastic hepatocytes with scattered mitotic figures form trabeculae with a disorganized architecture (B) and dysplastic hepatocytes adjacent to overt tumors show nuclear pleomorphism (left side of C). Only occasional HCC nuclei show β-catenin expression (arrows in F). Gastroenterology 2012 143, 1650-1659DOI: (10.1053/j.gastro.2012.08.047) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 Expression of liver cancer markers. Immunohistochemistry for GS, HSP70, AFP, and Ki-67 (all at 10×) in livers from animals 1 year after Cre induction. (Left) Axin1wt/Cre; (right) Axin1fl/fl/Cre mice bearing tumors. Arrows indicate positive staining in regions containing tumor. Gastroenterology 2012 143, 1650-1659DOI: (10.1053/j.gastro.2012.08.047) Copyright © 2012 AGA Institute Terms and Conditions