Fig 2: High-fat diet (HFD) feeding induces EPCAM+ biliary epithelial cell (BEC) proliferation.(A) Scheme depicting the isolation of EPCAM+ BECs from CD- and HFD-fed mice by fluorescence-activated cell sorting (FACS). (B) Gene set enrichment analysis (GSEA) of Gene Ontology (GO) terms. Top 15 upregulated biological processes (BP), ordered by normalized enrichment score (NES). q-value: false discovery rate adjusted p-values. (C–F) Representative co-staining images (C, E) and quantification (D, F) of BECs stained for OPN and Ki67 (C–D), or OPN and EdU (E–F) in livers of CD/HFD-fed mice. n=10 for Ki67 and n=5 for EdU. (G) Representative quantitative plots of the percentage (left) and quantification (right) of EdU+ EPCAM+ BECs, relative to panel A. n=5. (H) Schematic depicting BEC-organoid formation in vitro from CD/HFD-fed mouse livers. (I) Images of organoid colonies formed 6 days after seeding and quantification of organoids per well. n=5. Violin graphs depict the distribution of data points i.e the width of the shaded area represents the proportion of data located there. Other data are shown as mean ± SEM. **p<0.01; ****p<0.0001; unpaired, two-tailed Student’s t-test was used. PV, portal vein. Arrowheads mark bile ducts. Scale bars, 20 µm (C, E), 200 µm (I).

Fig 3: Heatmaps representing the expression of hepatocyte and BEC markers in EPCAM+ BECs isolated from livers of C57BL/6J mice fed with CD or HFD for 15 weeks (A) and BEC-organoids derived from the cells described in A (B).Heatmaps represent the Z-score calculated as “-1 * Z-score” from the expression data from RNAseq (A) and the d CT values from RT-qPCR (B), respectively. 36b4 was used as the housekeeping gene..