Fig. 4: DDC injury-induced ductular reaction causes proliferation of midlobular hepatocytes. | Nature Communications

Fig. 4: DDC injury-induced ductular reaction causes proliferation of midlobular hepatocytes.

From: Heterogeneity of hepatocyte dynamics restores liver architecture after chemical, physical or viral damage

Fig. 4

A Schematic showing the experimental design for the DDC-induced bile duct and hepatocyte injury model. Created with BioRender.com. B Images and quantification of Sirius red staining in liver sections from mice fed chow (control mice) or DDC diet for 6 weeks. Data are presented as mean values +/− SEM. 11 photos from 3 mice per group. Unpaired t test. CV central vein, PV portal vein. Scale bars, 100 μm. C Body weight (g), liver weight (mg) and spleen weight (mg) from mice fed chow or DDC diet for 6 weeks; Data are presented as mean values +/− SEM; n = 4 mice per diet. Unpaired t test. D Quantification of total number of cells by DAPI staining in mice fed chow or DDC diet for 6 weeks. Data are presented as mean values +/− SEM. 9 photos analyzed from 3 mice per group. Unpaired t test. E Quantification of the % for the positive area per field of view (10×) for CAG-EGFP in liver sections from mice fed chow and DDC diet for 6 weeks. Data are presented as mean values +/− SEM. 5–14 photos analyzed per mouse; n = 3 mice per group. Unpaired t test. F Representative CK19 and GS IHC analysis in liver sections from mice fed chow or DDC diet for 6 weeks (n = 4). Scale bars, 100 μm. G Liver sections from mice fed chow (left panels) or DDC diet (right panels) for 6 weeks. Top panels show CAG-EGFP and DAPI staining. Middle panels indicate the Rainbow fluorophores. Lower panels represent H&E staining showing a portal vein from both groups of mice. Asterisks indicate biliary porphyrin deposition. Scale bars, 100 μm. H Top graphs: quantification of number of cells per clone (y-axis) and number of clones (x-axis) per area of the liver lobule and per Rainbow fluorophore of liver sections from mice fed chow (control) or DDC diet for 6 weeks. Data are presented as mean values. 22–50 liver lobule areas analyzed from 3 mice per group (7–10 photos per mouse). One-way ANOVA, Tukey’s multiple comparisons post-test. Bottom graphs: relative frequency in % of each clone size per area of the liver lobule and per Rainbow fluorophore from mice fed chow (control) or DDC diet for 6 weeks. Data are presented as mean values +/− SEM. 22–50 liver lobule areas analyzed from 3 mice per group (7–10 photos per-mouse). I Ki67 IHC and TUNEL assay in liver sections from mice fed chow (control) or DDC diet for 6 weeks. Scale bars, 100 μm. J CD68 and CD45 IHC in liver sections from mice fed chow (control) or DDC diet for 6 weeks. Scale bars, 100 μm. K Quantification of the Ki67 IHC. Left graph: Total Ki67 positive cells (hepatocytes and non-hepatocyte cells) in % per field of view (10×) of liver sections from mice fed chow (control) or DDC diet for 6 weeks. 20 and 30 photos analyzed from 3 mice for control and DDC mice respectively. Right graph: proliferating Ki67 hepatocytes per field of view (10×) in each zone of the liver lobule from mice fed chow (control) or DDC diet for 6 weeks. 20 and 30 photos analyzed from 3 mice for control and DDC mice respectively. Data in left and right panels are presented as mean values +/− SEM. One-way ANOVA, Tukey’s multiple comparisons post-test. L Quantification of the TUNEL positive staining. Apoptosis area in % in liver sections from mice fed chow (control) or DDC diet for 6 weeks. 7–18 photos analyzed per mouse: 3 mice per group. Data are presented as mean values +/− SEM. Unpaired t test. M Quantification of the CD68 and CD45 positive staining. CD68 and CD45 area in % per field of view (10×) in liver sections from mice fed chow (control) or DDC diet for 6 weeks. CD68: Data are presented as mean values +/− SEM. 12–13 photos analyzed per mouse; 3 mice per group. Unpaired t test. CD45: Data are presented as mean values +/− SEM. 12–14 photos analyzed per mouse; 3 mice per group. Unpaired t test. N Hepatocyte ploidy in the liver lobule of mice fed DDC diet for 6 weeks. % of total polyploid and diploid hepatocytes within the liver lobule (left graph) and per area of the liver lobule (middle left graph). % of diploid hepatocytes (hepatocytes that express only 1 fluorophore) being mononucleated or multinucleated (middle right graph). % of polyploid hepatocytes (hepatocytes that express more than 1 fluorophore) being mononucleated or multinucleated (right graph). Data are presented as mean values +/− SEM. 5 photos analyzed per mouse; 3 mice. Source data are provided as a Source Data file.

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