Fig 2: RNA sequencing of organoids indicates loss of Pkd1 elevates Wnt/Ctnnb1 signaling.A Volcano plot showing distribution of genes differentially expressed in organoids of the Apc−/−;Pkd1−/− versus Apc−/− genotypes, based on statistical significance (Y axis, p value) and log2 fold change (x axis). B Waterfall plot demonstrating upstream transcriptional regulators statistically significantly changed in Apc−/−;Pkd1−/− versus Apc−/− organoids. C Expanded volcano plot focused on genes above cutoffs for statistical significance and magnitude of change, from A. Gene names shown reflect those linked to CTNNB1 signaling as targets identified by Ingenuity (23 of 30 total genes above cut-off values) or as regulators, based on literature (in italics). D, E Immunofluorescence staining of lesions from Apc−/−;Pkd1−/− versus Apc−/− mice colons for β-catenin (D), at 10x (left images) and 40x (right images), with quantification of signal intensity (E).

Fig 3: Linking β-catenin with SETD7-mediated LPM cell generation(A) GO enrichment of the upregulated genes in SETD7−/− #1 cells at day 2 of hematopoietic differentiation.(B) GSEA plot depicting the enrichment of the Wnt signaling pathway in WT and SETD7−/− #1 cells at day 2 of hematopoietic differentiation.(C–D) RT-qPCR assay showing the relative mRNA expression of designed genes in WT and SETD7−/− #1 cells at day 2 of differentiation. (n = 3, ∗p < 0.05, ∗∗p < 0.01, unpaired two-tailed Student’s t test). Data are means ± SD.(E) Western blot showing the protein levels of β-catenin and SETD7 in WT and SETD7−/− #1 hESCs. GAPDH was used as a loading control.(F) RT-qPCR assay representing the relative mRNA expression of CTNNB1 in WT and SETD7−/− #1 cells at day 2 of differentiation. (n = 3, ns, no significance, unpaired two-tailed Student’s t test). Data are means ± SD.(G) Western blot showing the expression of phosphorated β-catenin and SETD7 in nuclear and cytoplasmic of WT and SETD7−/− #1 cells at day 2 of differentiation. Lamin B was used as the nuclear loading control, and β-actin was used as the cytoplasmic loading control.(H) Coimmunoprecipitation of flag-tagged SETD7 and endogenous β-catenin in control (empty vector) and SETD7-overexpressing hESCs. Ten percent of the input was used as the total protein control.(I) Schematic illustration of the strategy used to construct the CTNNB1 K180R mutant expression vector.(J) Sanger sequencing confirmed the K180R mutation.(K) Western blot showing the expression of β-catenin and SETD7 in hESCs infected by the empty vector, CTNNB1 WT and CTNNB1 K180R-mutant vectors. GAPDH was used as the loading control.(L) Coimmunoprecipitation of β-catenin with anti-FLAG antibody and endogenous SETD7 in control (empty vector), CTNNB1 WT and CTNNB1 K180R mutant hESCs. Ten percent of the input was used as the total protein control. See also Figure S3 and Table S4.

Fig 4: Response to drugs targeting CFTR and CTNNB1, and chemotherapy, based on Pkd1 genotype.A, B Representative images (A) and quantitation (B) of organoid size following treatment with vehicle, the CFTR activator pair lumacaftor/ivacaftor (L/I), or the CTNNB1 inhibitor PRI-724 (PRI), based on Apc−/− versus Apc−/−;Pkd1−/− genotype. Scale bars on (A), 50 μm. C Experimental design to assess in vivo response to FOLFIRI or FOLFOX. D, E Representative image of colons from mice treated with FOLFOX and FOLFIRI; gross presentation (D) and H&E-stained tissue (E). F, G Quantification of number of lesions based on gross assessment of number in colon (F) and area, based on histopathological assessment (G) following treatment with vehicle, FOLFIRI, or FOLFOX, in genotypes indicated. H Quantitation of viability based on CellTiterGlo of organoid cultures following treatment with vehicle, FOLFIRI, or FOLFOX, in indicated genotypes, with data normalized to vehicle-treated Apc−/− organoids. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 for all graphs; in (B), ###p < 0.001; compared to Apc−/− vehicle.

Fig 5: Ph-S675-β-catenin is increased in patients with aggressive HBL and in HBL cell lines HepG2 and Huh6. (A) Structure of the CEGRs/ALCDs in cancer genes is shown. Black box shows TCF4 binding sites in the CEGRs/ALCDs of β-catenin, TCF4, and AFP genes. (B) Levels of CTNNB1 (β-catenin) mRNA in fresh HBL biobank (n = 32) (p = 0.0127, unpaired t-test) (C) Levels of total β-catenin and ph-S675-β-catenin in background and tumor regions of HBL. Bar graph shows levels of ph-S675-β-catenin as a ratio to β-actin (p = 0.0017, unpaired t-test). (** p ≤ 0.05) (D) IF staining of livers of HBL patients with antibodies to ph-S675-β-catenin and stained with DAPI. Arrows indicate nuclear localization of ph-S675-β-catenin. (E) ph-S675-β-catenin is elevated in mitotic (shown by arrows) Huh6 and HepG2 cells. The uncropped Western blots have been shown in Figure S9.