Fig 2: Histological features of ANIT-induced cholestasis model. (A) H&E and Sirius red stained liver sections from normal group, 8w model group, 12w model group and YCHT-treated group. Normal, healthy rats; 8w Model, ANIT treatment for 8 weeks; 12w Model, ANIT treatment for 12 weeks; YCHT-treated, ANIT-induced cholestasis rats treated with YCHT from 9th week to 12th week. Original magnifications × 100 for H&E, and × 200 for Sirius red. (B) Immunofluorescence analysis of CK19 in liver sections. (C) The positive area of CK19 in different groups. (D) Immunofluorescence analysis of Collagen I in liver sections. (E) The positive area of Collagen I in different groups. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 3: Myofibroblasts induce the conversion of mature hepatocytes into ductal biliary epithelial cells in vivo.a Experiment design for DAPT-mediated inhibition of HSC activation in TAA-treated RosaYFP reporter mice. b Immunofluorescent staining of fibrotic liver from TAA-treated RosaYFP reporter mice. Immunostaining of ductal BECs marker (CK19, OPN, and SOX9) in liver of RosaYFP mice showed that the percentage of hepatocytes-derived ductal BECs was diminished after DAPT-mediated inhibition of HSC activation in TAA-treated mice. c Experiment design for DAPT-mediated inhibition of HSC activation in CCl4-treated RosaYFP reporter mice. d Immunofluorescence staining of fibrotic liver from CCl4-treated RosaYFP reporter mice. e Experiment design for DAPT-mediated inhibition of HSC activation in DEN/CCl4-treated RosaYFP reporter mice. f Immunofluorescence staining of fibrotic liver from DEN/CCl4-treated RosaYFP reporter mice. Magnification, ×600.

Fig 4: Numb knockdown reduces the inhibitory effect of HQD on WB-F344 differentiation into cholangiocyte phenotype. (a) Lentivirus transfected WB-F344. (b) Relative Numb level measured by qRT-PCR. (c) Relative CK19 level measured by qRT-PCR. All mRNA levels were normalized by GAPDH (n = 3 per group). (d) Immunostaining of CK19 (×600). *P < 0.05, **P < 0.01. N, normal group; SB, sodium butyrate group; HQD, SB plus Huang Qi Decoction group; DAPT, SB with DAPT group; NC, empty virus with SB group; Numb-KD, Numb knockdown with SB group; and HQD + Numb-KD, Numb knockdown and SB with Huang Qi Decoction group.

Fig 5: Immunofluorescence identification of epithelial marker keratin antibody was performed on endometrial cells and oviduct mucosa cells of the second generation. The results showed that 80–90% of the cytoplasm of endometrial cells and oviduct mucosa cells were stained with green fluorescence, showing positive cytokeratin antibodies. Microscopically, the cells were polygonal, spindle and spindle. Immunofluorescence identification of interstitial cell marker vimentin antibody was performed on endometrial cells and oviduct mucosa cells of the fifth generation. 80% of the cytoplasm of endometrial cells and oviduct mucosa cells were stained with red fluorescence under microscope. It showed positive cell vimentin antibody, and the cells were fusiform and polygonal under the microscope. (A1) The second generation of endometrial cells (DAPI). (A2) The second generation of endometrial cells (CK19). (A3) The second generation of endometrial cells (Merge). (B1) The second generation of oviduct mucosal cells (DAPI). (B2) The second generation of oviduct mucosal cells (CK19). (B3) The second generation of oviduct mucosal cells (Merge). (C1) The fifth generation of endometrial cells (DAPI). (C2) The fifth generation of endometrial cells (Vimentin). (C3) The fifth generation of endometrial cells (Merge). (D1) The fifth generation of oviduct mucosal cells (DAPI). (D2) The fifth generation of oviduct mucosal cells (Vimentin). (D3) The fifth generation of oviduct mucosal cells (Merge).