Volume 42, Issue 3, Pages 378-385 (March 2005) Oncosis represents the main type of cell death in mouse models of cholestasis  Peter Fickert, Michael Trauner, Andrea Fuchsbichler, Gernot Zollner, Martin Wagner, Hanns-Ulrich Marschall, Kurt Zatloukal, Helmut Denk  Journal of Hepatology  Volume 42, Issue 3, Pages 378-385 (March 2005) DOI: 10.1016/j.jhep.2004.10.016 Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Jo2-treated mice: the classical rodent model for hepatocyte apoptosis.(A) In Fas-activated mice (0.25μg/g BW i.p. injection of the Fas agonist Jo2) numerous hepatocytes show characteristic signs of apoptosis (i.e. pyknotic nuclei, condensation of chromatin, hepatocytes shrink with dense cytoplasm) (arrow heads). (B) Activated caspase-3 (red) is detectable in several hepatocytes (asterisks). (C) Double-fluorescence labeling for CKs (red) and TUNEL (green) reveals CK-IF breakdown and cytoplasmic condensation of CK 18 (characteristic of apoptotic epithelial cell death) (asterisks) in TUNEL-positive shrunken hepatocytes (arrow heads). (D) Double-immunofluorescence labeling for CK 18 (green) and activated caspase-3 (red, due to colocalization yellow in the merged figure) reveals caspase-3-positive granules of condensed, cleaved CK 18. Original magnification for A×20 and B×40. Bar for C and D is 20μm. Journal of Hepatology 2005 42, 378-385DOI: (10.1016/j.jhep.2004.10.016) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 2 False-positive TUNEL assay in CA-fed mouse liver. (A) CK-IF breakdown and cytoplasmic condensation of CK 18 (characteristic of apoptotic epithelial cell death) in Fas-activated (Jo2) mouse liver (asterisks). (B) TUNEL-positive shrunken nuclei (indicated by arrow heads). (C) Double-fluorescence labeling reveals correlation between classical apoptotic features of the CK-IF network and cells with positive TUNEL staining. (D) Swollen hepatocyte with preserved CK-IF network in CA-fed mouse liver (asterisk) and (E) positive TUNEL staining (arrow heads). (F) TUNEL-positive cell lacking CK alterations typical for apoptosis. Bar is 10μm. Journal of Hepatology 2005 42, 378-385DOI: (10.1016/j.jhep.2004.10.016) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Ultrastructural characteristics of hepatocytes in Fas-activated and cholic acid (CA)-fed mouse liver. (A) Fas-activated liver showing shrinkage of a dying hepatocyte with chromatin condensation in the nucleus (N). (B) Higher magnification showing chromatin condensation and intact nuclear membrane (N) and characteristic apoptotic lamellar body (myeline figure; asterisk). (C) Oncotic hepatocyte in CA-fed mouse liver with cell edema and chromatin clumping in the nucleus (N). (D) Higher magnification showing ruptured mitochondria (M; indicated by arrow heads) and nuclear membrane. Original magnification for A, C×1650, for B, D×8900. Journal of Hepatology 2005 42, 378-385DOI: (10.1016/j.jhep.2004.10.016) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Analyis of DNA ladder by agarose gel electrophoresis under different experimental conditions. DNA was extracted from livers and 3μg were run on agarose gels as described in Section 2. Lanes: (1) standard DNA 100bp ladder, (2) standard diet-fed control, (3) Jo2-treated mouse liver, (4) CA-fed mouse liver, (5) sham-operated control, (6) CBDL mouse liver. Note DNA laddering (indicative for apoptotic cell death) only in the Jo2-challenged mouse liver. Journal of Hepatology 2005 42, 378-385DOI: (10.1016/j.jhep.2004.10.016) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Oncosis is the main type of cell death in CA-fed mice. (A) In these livers numerous swollen pale-staining hepatocytes (arrow heads; H and E staining) with loss of nuclear staining are present, indicative of disseminated hepatocellular oncosis leading to necrosis. (B) Oncotic hepatocytes (asterisks) lack staining for activated caspase-3 (red). Positive staining for activated caspase-3 (arrow head) in hepatocytes is a very rare event. (C) Double-fluorescence labeling for CKs (red) and TUNEL (green) reveals a diffuse pattern of the CK-IF network in TUNEL-positive swollen hepatocytes. (D) Double-immunofluorescence labeling for CK 18 (green) and activated caspase-3 (red) reveals negative staining for caspase-3. Note also the lack of CK-IF network condensation/breakdown in oncotic hepatocytes (asterisks). Cv, central vein. Original magnification for A×20 and B×40. Bar for C and D is 20μm. Journal of Hepatology 2005 42, 378-385DOI: (10.1016/j.jhep.2004.10.016) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Oncosis represents the main type of cell death in common bile duct ligated (CBDL) mice. (A) In CBDL mice numerous swollen hepatocytes constituting a characteristic bile infarct (arrow heads) show loss of nuclear staining indicative of hepatocellular oncosis leading to necrosis. In addition, the necrotic area is infiltrated by some neutrophils (H and E). (B) The area of the bile infarct/oncotic hepatocytes (border indicated by arrow heads) mostly lacks immunostaining for activated caspase-3 (red). Positive activated caspase-3 staining (arrow) of hepatocytes is a very rare event. (C) Immunofluorescence labeling for CK 18 (green) reveals a diffuse pattern of the CK-IF network in swollen hepatocytes in a bile infarct. (D) In addition, double-immunofluorescence labeling for CK 18 (green) and activated caspase-3 (red) reveals that most damaged hepatocytes lack staining for activated caspase-3. n, necrosis (bile infarct); bd, bile duct. Original magnification for A and B×20. Bar is 50μm. Journal of Hepatology 2005 42, 378-385DOI: (10.1016/j.jhep.2004.10.016) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions