IKK2-deficient hepatocytes are more sensitive to bile acid induced apoptosis compared to wild type cells.

<p>(A) Representative images of DAPI stained primary WT and IKK2-KO hepatocyte cultures. Cells were left untreated or were stimulated with taurolithocholic acid 3-sulfate disodium salt (TLCS, 100 µM) for the indicated time periods. Arrowheads show apoptotic hepatocytes as indicated by smaller highly fluorescent nuclei with condensed chromatin. Scale bars, 100 µm. (B) Graphs showing the average proportion of apoptotic hepatocytes from 3 independent experiments. Mean values ± SEM shown. * p<0,05; ** p<0,01; n.s.: not statistically significant. (C) Immunoblot detection of cleaved Caspase-3. Primary hepatocytes were stimulated with glycochenodeoxy-cholate (GCDC, 50 µM, top) or TLCS (100 µM, bottom) for the indicated time periods. Tubulin serves as loading control.</p

Mdr2^−/−IKK2^Hep-KO mice show impaired growth and signs of jaundice.

<p>(A) Immunoblot of whole liver lysates from 3 representative mice for each depicted genotype. Tubulin serves as loading control. (B) Weight curve of male mice for the denoted genotypes. * p<0,05 for Mdr2^+/− IKK2^Hep-KO vs. Mdr2^−/−, ** p<0,001 for Mdr2^−/− IKK2^Hep-KO vs. Mdr2^−/−. (C) Macroscopic appearance of littermate male mice at 12 weeks of age. Genotypes as specified in the picture. (D) Necropsy of littermate mice from (C). White arrowheads: enlarged gall bladder in double knockout mouse. Black arrowhead: jaundiced peritoneal serosa in double knockout mouse vs. normal appearance of serosa in Mdr2^−/− and Mdr2^+/−IKK2^Hep-KO mice. The ruler indicates centimeters in C and D.</p

Mdr2^−/−IKK2^Hep-KO mice exhibit increased liver damage, hepatocyte death and cholestasis.

<p>(A) Alanine aminotransferase (ALT), (B) alkaline phosphatase (AP), and (C) bilirubin levels in serum of adult male mice (8–26 weeks) with the indicated genotypes. Error bars indicate SEM. Number of mice: Mdr2^−/−IKK2^Hep−KO: n = 10, Mdr2^−/−: n = 7, Mdr2^+/−IKK2^Hep-KO: n = 7 and Mdr2^+/+IKK2^FL: n = 2. (D–E) Representative pictures of TUNEL (D) and PCNA staining (E) on sections of paraffin-embedded livers from mice with the indicated genotypes. The green signal in E corresponds to background auto-fluorescence and was used to visualize the general morphology of the liver tissues. Scale bars: 20 µm.</p

Mdr2^−/−IKK2^Hep-KO mice develop severe fibrosis.

<p>(A) Representative images of Masson trichrome stained livers from 12-week-old littermate male mice and from 27–30 week-old male mice. (B) Representative images of Sirius Red stained livers from 12-week-old littermate male mice and from old adult male mice (27–30 weeks). Insert: High magnification of corresponding liver sections. Arrowhead: Pericellular fibers. Double arrowhead: Pericellular space free of stained fibers. Genotypes as in (A). (C) Quantification of fibrosis in mice with the depicted genotypes measured as percentage of Sirius Red positive area as a fraction of the total area of at least 10 high power fields per mouse. Left: young adult mice (males, 8–19 weeks), right: old adult mice (males, 20–42 weeks). Error bars indicate SEM. Scale bars, 100 µm.</p

Histological analysis of the liver pathology in Mdr2^−/−IKK2^Hep-KO mice.

<p>(A) Representative images of Hematoxylin & Eosin (HE) stained livers from young (12-week-old) littermate male mice and old (27–30 weeks) mice with the specified genotypes. Insert: High magnification of corresponding liver sections. (B) Immunofluorescence staining of cytokeratin 19 (CK19) in old male mice. DNA was stained with DAPI. The dashed line marks the distance from one portal field to the next, showing extreme abundance of CK19 positive cells in the Mdr2^−/− IKK2^Hep-KO as compared to Mdr2^−/− or Mdr2^+/−IKK2^Hep-KO mice. PV portal vein. CV central vein. BD bile duct. Scale bars, 100 µm.</p

JHC768230_Supplemental_Material – Supplemental material for Depolarized Hepatocytes Express the Stem/Progenitor Cell Marker Neighbor of Punc E11 After Bile Duct Ligation in Mice

<p>Supplemental material, JHC768230_Supplemental_Material for Depolarized Hepatocytes Express the Stem/Progenitor Cell Marker Neighbor of Punc E11 After Bile Duct Ligation in Mice by Andrea Bowe, Susanne Zweerink, Vera Mück, Vangelis Kondylis, Sigrid Schulte, Tobias Goeser and Dirk Nierhoff in Journal of Histochemistry & Cytochemistry</p