Fig 2: SB, KPPC, and FAC mouse tumors have phenotypic features of CCA. (A) Representative photomicrographs of H&E-stained (left panel) and KRT19 immunohistochemistry (IHC) (right panel) of SB tumor sections from WT and Trail-r-/- mice. Scale bar: 50 μm. (B) Representative photomicrographs of H&E (left panel), pan-cytokeratin (Pan-CK) and hepatocyte nuclear factor 4α (HNF4α) IHC (middle panels), and immunofluorescence depicting costaining of pan-CK and HNF4α (right panel) of KPPC tumor sections from WT and Trail-r-/- mice. Nuclei counterstained with Hoechst stain in immunofluorescence images. Scale bar: 50 μm. (C) Representative photomicrographs of H&E-stained (left panel) and KRT19 IHC (right panel) of FAC tumor sections from WT mice. Scale bar: 50 μm. All photomicrographs represent results from at least n ≥ 3 mice.

Fig 3: Tamoxifen treatment decreases protein expression of αSMA and KRT19. Mice were fed standard or DDC-supplemented diet, and treated with tamoxifen or vehicle, as indicated. Liver sections were first incubated with anti-αSMA or anti-KRT19 antibody as indicated, and then incubated with HRP-conjugated secondary antibody and stained with diaminobenzidine (DAB). Standard diet-fed mice had low expression of αSMA and KRT19 regardless of treatment. DDC-feeding increased expression of αSMA and KRT19 in vehicle-treated mice, as shown by brown staining and tamoxifen treatment significantly reduced this increase. Sections are from male mice, and the same pattern was obtained in female mice (not shown). The anti-keratin antibody stains both keratin 17 and keratin 19, but in liver, keratin 19 is predominant. αSMA—alpha smooth muscle actin, Krt19—keratin 19, TMX—tamoxifen, DDC—3,5-diethoxycarbonyl-1,4-dihydrocollidine.

Fig 4: EZH2-mediated miR-22 suppression promotes proliferation and differentiation of HFSCs. (A) The expression of miR-22, and mRNA expression of STK40, MEF2 and ALP in HFSCs of EZH2-/- mice or WT mice determined by RT-qPCR. (B) Protein expression of STK40, MEF2, ALP, differentiation-related proteins (β-catenin, TCF-4), and TA cell differentiation markers (CK15, CK19) in HFSCs of EZH2-/- mice or WT mice normalized to GAPDH determined by Western blot analysis. (C) Colony forming capacity of HFSCs from EZH2-/- mice or WT mice determined by colony formation assay. (D) The proliferation and viability from EZH2-/- mice or WT mice evaluated by MTS assay. (E) HFSCs from EZH2-/- mice or WT mice at different cell phases observed by flow cytometry. * p < 0.05 vs. WT mice; # p < 0.05 vs. NC-inhibitor (EZH2-/-) group; Measurement data were expressed as mean ± standard deviation. One-way ANOVA was utilized to compare data among multiple groups, followed by Tukey’s post hoc test. Repeated measures ANOVA was adopted to analyze data among multiple groups at different time points, followed by Bonferroni posttest. Cell experiments were conducted in triplicates.

Fig 5: miR-22 inhibits proliferation and differentiation of HFSCs by downregulating STK40 and suppressing MEF2-ALP activity. (A) Protein expression of STK40, MEF2, ALP, differentiation-related proteins (β-catenin, TCF-4), and TA cell differentiation markers (CK15, CK19) in HFSCs normalized to GAPDH determined by Western blot analysis. (B) Colony forming capacity of HFSCs determined by colony formation assay. (C) HFSC proliferation and viability evaluated by MTS assay. (D) HFSC cell cycle changes revealed by flow cytometry. * p < 0.05 vs. NC-inhibitor group; # p < 0.05 vs. miR-22-inhibitor + si-NC group; Measurement data were expressed as mean ± standard deviation. One-way ANOVA was utilized to compare data among multiple groups, followed by Tukey’s post hoc test. Repeated measures ANOVA was adopted to analyze data among multiple groups at different time points, followed by Bonferroni posttest. Cell experiments were conducted 3 times independently.