Depletion of Cd34 ⁺ cells affected ductal homeostasis and tumorigenesis. (A) Confocal images of liver sections from 8-wk-old Cd34^creER;R26^tdTomato and Cd34^creER;R26^DTA/tdTomato mice that had been treated with tamoxifen at 6 wk of age. Scale bar = 100 µm. (B) Numbers of Tomato⁺ cells in Cd34^creER;R26^DTA/tdTomato liver sections were quantified relative to the numbers of Tomato⁺ cells in Cd34^creER;R26^tdTomato liver sections (n = 5 mice per genotype from three independent experiments). (C and D) Anti-Sox9 staining of paraffin liver sections (C) from 8-wk-old R26^DTA and Cd34^creER;R26^DTA mice treated with tamoxifen at 6 wk of age. Scale bar = 50 µm. The numbers of Sox9⁺ cholangiocytes forming the circumference of the bile ducts were quantified (D) (n = 5 mice per genotype from three independent experiments). (E and F) EdU incorporation of R26^DTA and Cd34^creER;R26^DTA mice in a period of 4 wk right after tamoxifen administration. Cholangiocytes were revealed by anti-Epcam staining (E). The percentages of all DAPI⁺ cells and DAPI⁺Epcam⁺ cells that incorporated EdU were quantified (F). Scale bar = 25 µm (n = 5 mice per genotype from three independent experiments). (G and H) Sirius red staining of paraffin liver sections (G) from DDC-treated R26^DTA and Cd34^creER;R26^DTA mice (pretreated with tamoxifen at 6 wk of age). DDC treatment was started at 8 wk of age. Scale bar = 100 µm. The percentage of Sirius red–stained tissue within liver sections was quantified (H) (n = 5 mice per genotype from three independent experiments). (I and J) Relative mRNA levels of Col1a1 (I) and Acta2 (J) in non-parenchymal liver cells from R26^DTA and Cd34^creER;R26^DTA mice under normal conditions or after DDC treatment. The mRNA level in R26^DTA mice under normal conditions was set as 1. Data represent the mean ± SD (n = 5 mice per genotype from three independent experiments). (K and L) Confocal images of liver sections from DDC-treated Cd34^creER;R26^tdTomato and Cd34^creER;R26^tdTomato;Tgfbr2^fl/fl mice, stained with DAPI and anti-Col1 antibody (K). Scale bar = 50 µm. Data represent the mean ± SD (L) (n = 5 mice per genotype from three independent experiments). (M–P) H&E staining of paraffin liver sections (M and O) from ICC-bearing R26^DTA and Cd34^creER;R26^DTA mice (pretreated with tamoxifen at 6 wk of age). ICC was induced at 8 wk of age. Scale bar = 200 µm. The percentage of tumor area within liver sections was quantified (N and P) (n = 5 mice per genotype from three independent experiments). (Q and R) Relative mRNA levels of Col1a1 (Q) and Acta2 (R) in non-parenchymal liver cells from R26^DTA and Cd34^creER;R26^DTA mice under normal conditions or after ICC induction. The mRNA level in R26^DTA mice under normal conditions was set as 1. Data represent the mean ± SD (n = 5 mice per condition from three independent experiments). The statistical significance of differences was always assessed by two-tailed Student’s t tests. *P < 0.05, **P < 0.01, ***P < 0.001.