Fig 1: Loss of CAR accelerates cholestatic liver injury. (A) BW changes of WT, DKO, and TKO mice (n = 3-5). (B) Representative image of whole body and liver at 3 months old. Scale bar, 10 mm; arrowhead, cholecystomegaly. (C) BW, liver weight, liver/BW ratio (%) (n = 8), and (D) gallbladder volume (n = 5) were measured. (E) Hematoxylin and eosin staining (magnification ×100) and KRT19 immunostaining (magnification ×40). ANOVA followed by Tukey HSD; #P < 0.05, ###P < 0.005. Abbreviations: GB, gallbladder; H&E, hematoxylin and eosin; LW, liver weight.
Fig 2: Characterization of WLC, HC and NPC spheroids. (A) Hematoxylin and Eosin (H&E) staining and CD68, aSMA, HNF4A and CK19 immunohistochemistry (IHC) of Day 7 WLC, HC and NPC spheroids for the indicated hepatic lineage markers. Scale bar: 100 µm. (B) Quantitative RT-PCR of Day 7 WLC, HC and NPC spheroids for the two indicated HC marker genes. Table below shows the unpaired two-tailed Student’s t-test comparison of the indicated culture conditions; *P<0.05, ***P<0.001, ****P<0.0001. (C) Albumin IHC and periodic acid–Schiff (PAS) staining of WLC spheroids (SphWLC) and HC spheroids (SphHC). Scale bar: 50 µm. (D) Albumin levels in the spheroid conditioned medium measured by enzyme-linked immunosorbent assay on Day 1, 4 and 7. A base level of albumin was detected in NPC spheroids (SphNPC) because a 0.1% bovine serum albumin solution was used to dissolve the growth factors supplemented in the spheroid culture medium. (E) Low-density lipoprotein (LDL) uptake assay of the spheroids with (+) or without (-) anti-LDL antibody (LDL-Ab). Scale bar: 50 µm.
Fig 3: (A) qRT-PCR analysis from total RNA in liver after irradiation (25 Gy) with and without TNF-a at various time-points (1-48 hours), compared to sham-irradiated controls (Co.), which received both PBS (i.p.) and sham-irradiation. Fold-change in mRNA expression of CD68 is shown. qRT-PCR was normalized using two housekeeping genes: ß-actin and GAPDH. Results represent mean values ± SEM of each five animals. Note the significant effects of TNF-a after 6 and 12 hours. * = Comparison irradiation (25 Gy) vs irradiation + TNF-a treatment. (B-H) Detection of CK-19 (green, marker for biliary cells) and F4/80 (red, marker for macrophages) by double immunofluorescence staining in murine liver after irradiation (25 Gy) in the presence/absence of TNF-a, compared to sham-irradiated controls. (B) Liver of control mice; (C) 6 hours, (D) 12 hours, (E) 24 hours after irradiation. (F) 6 hours, (G) 12 hours, (H) 24 hours after TNF-a plus irradiation. Nuclei were stained with DAPI (blue). Note the significantly larger amounts of macrophages in TNF-a-treated specimens, which is most obvious after 6 and 12 hours. Original magnification ×100
Fig 4: Decrease of liver biliary network density in the Lyve1-Cre;Hegfl/flmice.A-B, 3-D visualization of biliary network (A) and quantitation of peripheral biliary branches (B) in the liver of Lyve1-Cre;Hegfl/fl and control mice at 4, 8 and 24 wk of age. C-D, Immunostaining of CK19 (C) and quantitative plots of total and portal CK19 counts (D) indicating the bile duct density and bile duct/PV ratio in Lyve1-Cre;Hegfl/fl and control mice at 3 weeks of age. E-F, Immunostainings of Podoplanin (Pdpn) and CK19 (E) and quantitative plots of the number of Pdpn+ lymphatic vessels (F) show no obvious defect in periportal lymphatic vessels in the liver of Lyve1-Cre;Hegfl/fl mice at 8 weeks. **P < .01; ***P < .001; Scale bars represent 5 mm in panel A, 100 µm in panel C, and 50 µm in panel E.
Fig 5: High-dose tamoxifen administration does not induce pancreatitis.(A–D) Lowand (A'–D') high-magnification H&E staining of wild-type pancreata, fixed 24 hr after indicated treatment. (E–H) Immunofluorescence for CK19 (red), CD45 (green) and DAPI (blue), highlighting inflammatory cells in the exocrine pancreas. Scale bars: (A–D) 200 µm, (A'–D') 100 µm, (E–H) 50 µm.DOI: http://dx.doi.org/10.7554/eLife.07125.015
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