The BH3-Only Protein Bid Does Not Mediate Death-Receptor-Induced Liver Injury in Obstructive Cholestasis  Padmavathi devi Nalapareddy, Sven Schüngel, Ji-Young Hong, Michael P. Manns, Hartmut Jaeschke, Arndt Vogel  The American Journal of Pathology  Volume 175, Issue 3, Pages 1077-1085 (September 2009) DOI: 10.2353/ajpath.2009.090304 Copyright © 2009 American Society for Investigative Pathology Terms and Conditions

Figure 1 A: Wild-type (WT) and Bid−/− mice underwent BDL and were euthanized after 3, 7, and 14 days. Serum levels of AST and bilirubin as markers of hepatic damage were measured at indicated time points. Mean values and SD are presented for all time points (n = five in each group). B: H&E staining revealed profuse liver damage and multiple oncotic bile infarcts in BDL mice. C: Survival of WT and Bid−/− mice was recorded after BDL (n = 20 mice in each group). The American Journal of Pathology 2009 175, 1077-1085DOI: (10.2353/ajpath.2009.090304) Copyright © 2009 American Society for Investigative Pathology Terms and Conditions

Figure 2 A: TUNEL and cleaved caspase-3 staining of liver sections is shown from wild-type (WT) and Bid−/− mice after BDL and in mice injected with the Fas mAb Jo-2 (original magnification, ×200, n = four in each group). B: Hepatic caspase-3 activity was measured by using the CaspACE Assay kit and processing of caspase-9 (loss of full length caspase-9) and caspase-3 (detection of the cleaved fragment) was analyzed by Western blot for all time points before and after BDL and in Jo-2 injected mice as a positive control (+). C: Liver tissues of control and BDL mice were analyzed for total cellular levels of Fas, Flip, and Bid (n = 5, pooled samples). The American Journal of Pathology 2009 175, 1077-1085DOI: (10.2353/ajpath.2009.090304) Copyright © 2009 American Society for Investigative Pathology Terms and Conditions

Figure 3 A, B: Hepatocyte proliferation was assessed with BrdU immunostaining and BrdU positive cells were quantified (10 microscope fields at ×200 magnification, wild-type (WT) mice are represented as open bars and Bid−/− mice as closed bars, n = 5). C: Immunoblots were performed for cyclin D and E and p21 (n = 5, pooled samples). The American Journal of Pathology 2009 175, 1077-1085DOI: (10.2353/ajpath.2009.090304) Copyright © 2009 American Society for Investigative Pathology Terms and Conditions

Figure 4 A: Liver fibrosis was evaluated by α-SMA, Sirius red, and Masson’s trichrome staining 3, 7, and 14 days after BDL (Original magnification, ×200). B: Hydroxyproline content measured in the right median (#3) and the right lateral (#5) liver lobes 7 days after BDL. C: mRNA levels of transforming growth factor-β, α-SMA, and matrix metalloproteinase-3 were quantified by real-time RT PCR (sham operated mice are represented with open bars and BDL mice with closed bars). The American Journal of Pathology 2009 175, 1077-1085DOI: (10.2353/ajpath.2009.090304) Copyright © 2009 American Society for Investigative Pathology Terms and Conditions

Figure 5 A: Liver sections of control and BDL mice were analyzed for CD68 expression and CD11b expression (Original magnification, ×200). Infiltrating neutrophils were also detected by (arrows) Naphthol ASD staining. B: Quantitative RT-PCR was performed by using pooled RNA extracted from wild-type (WT) and Bid−/− mice before (open bars) and after BDL (closed bars) (n = 4). Specific cDNAs of KC, monocyte chemotactic protein-1, interleukin-6, and TNF-α were amplified. The American Journal of Pathology 2009 175, 1077-1085DOI: (10.2353/ajpath.2009.090304) Copyright © 2009 American Society for Investigative Pathology Terms and Conditions

Figure 6 A: Wild-type (WT) and Lpr mice underwent BDL and were euthanized after 3 days (n = 4). Liver injury was analyzed by H&E staining. B: Western blot of p-Erk, p-Jnk, p-p38, and c-jun in WT and Bid−/− mice before and 3, 7, and 14 days after BDL. C: Analysis of p-Erk, p-Jnk, p-p38, and c-jun in WT and Lpr mice before and 3 days after BDL (positive control (+) for p-Jnk: WT mouse injected with Jo-2; positive control (+) for p-p38: Fah−/− mouse injected with homogentisic acid). D: Representative H&E staining of control and c-jun−/− mice 3 days after BDL. E: Serum AST and bilirubin levels were determined before and 3 days after BDL. The American Journal of Pathology 2009 175, 1077-1085DOI: (10.2353/ajpath.2009.090304) Copyright © 2009 American Society for Investigative Pathology Terms and Conditions