Fig 2: Sox9 promotes biliary identity and limits TGF-β signaling in BECs.a Bulk RNA-seq identifies differentially expressed genes between Sox9cKO and control BECs. Differential expression (DE) analysis was performed, and genes with a log fold change ≥1 and a q < 0.05 were considered statistically significant. b GSEA demonstrates significant enrichment of TGF-β signaling in Sox9cKO BECs relative to control BECs. c Co-localization of pSMAD2 and EpCAM confirms elevated TGF-β signaling in Sox9cKO BECs (scale bar represents 5μm; white asterisks indicate portal vein). d Targeted DE analysis of TGF-β receptor expression in bulk RNA-seq reveals that Tgfbr2 is the only significantly upregulated receptor and Amhr2 is the only downregulated receptor (asterisk indicates significance, p < 0.05). e Targeted DE analysis of TGF-β ligand expression in bulk RNA-seq reveals that the only significantly upregulated ligand is Inhba (TGF-β ligands that are not detected are not shown: Bmp2, Bmp15, Nodal, Gdf3, and Gdf8; asterisk indicates significance, p < 0.05). f IF demonstrates elevated Activin A in EpCAM+ BECs in Sox9cKO livers (scale bar represents 50 μm; white asterisks indicate portal vein). g GSEA demonstrates that Sox9cKO BECs are de-enriched for adult BEC gene signature (NES: −2.70; p = 3.67E-10) and enriched for adult hepatocyte gene signature (NES: 2.31; p = 3.67E-10). h GSEA of liver development gene sets shows that the Sox9cKO BEC transcriptome resembles embryonic BECs (NES: 1.23; p = 6.06E-6) and embryonic hepatocytes (NES: 2.32; p = 3.00E-10), but not hepatoblasts (NES: 0.99; p = 0.55). i Co-localization of NCAM1 and EpCAM is significantly elevated in Sox9cKO compared to controls (scale bar represents 5 μm; white asterisks indicate portal vein; p < 0.5, unpaired two-sided t-test; data are representated as). a-i, n = 3 biological replicates per group. b, g, and h gene set enrichment analysis was performed, and a p < 0.5 was considered significant. Source data are provided as a Source Data file.