Extended Data Fig. 4: R26^LSL-MYC liver is apoptotic and Ctnnb1^ex3/WT mutation uniformly activates the Wnt pathway across the liver lobule.

a, Representative images of cPARP immunohistochemistry, n = 7. b, c, Quantification of apoptotic cells by IHC for cleaved PARP (cPARP) and cleaved caspase-3 (CC3) 10 days post AAV8.TBG.Cre induction. b, Biological replicates: WT n = 8, R26^LSL-MYC (M) n = 10, Ctnnb1^ex3/WT (B) n = 7, Ctnnb1^ex3/WT;R26^LSL-MYC (BM) n = 11. Bars are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons test. c, Biological replicates: WT n = 9, B n = 11, M n = 10, BM n = 9. Bars are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons test. d, Apoptosis gene set enrichment in differentially expressed genes among TCGA-LIHC binned by CTNNB1 mutation status and MYC expression. A two sided, pairwise Wilcoxon test was used to compare data to the MYChigh/CTNNB1:WT group. Boxplots represent the median (central line) and 25th and 75th percentiles of the data (box). Whiskers represent the maximum and minimum of non-outlier values within 1.5× the interquartile range. Data beyond the whiskers are outliers. N = 372. e-g, NanoString GeoMx digital spatial profiling of Ctnnb1^ex3/WT day-4 liver. e, Representative image of immunofluorescence masks used to define the regions selected for spatial transcriptomics. BrdU (red), GLUL (yellow), DNA (blue). PN, portal node, adj., adjacent. f, Principal component analysis (PCA) of spatial transcriptomics data. ROI per Biological replicate: n = 8. g, Gene set variance analysis (GSVA) scores for liver-zonation gene sets Lobule layers (L1–9), CV, layer 1 to PN, layer 9 according to Halpern et al.²⁵. Biological replicates n = 4. h, Representative CTNNB1 IHC in AAV8.TBG.Cre-treated WT and Ctnnb1^ex3/WT liver at day 4 post induction, n = 3. Panels 1 - 4 are higher magnification images of the dashed areas in the left panel, highlighting hepatocytes near the PN and the CV. All scale bars, 100 μm.

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