Volume 132, Issue 7, Pages 2504-2517 (June 2007) Hepatocyte-Specific IKKγ/NEMO Expression Determines the Degree of Liver Injury  Naiara Beraza, Tom Lüdde, Ulrike Assmus, Tania Roskams, Sara Vander Borght, Christian Trautwein  Gastroenterology  Volume 132, Issue 7, Pages 2504-2517 (June 2007) DOI: 10.1053/j.gastro.2007.03.045 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Generation of hepatocyte-specific conditional ko of NEMO in mice by setting the Cre recombinase under the control of an albumin promoter. (A) Genomic DNA was analyzed by Southern blot. A 3.2-kilobase band evidences that NEMO gene is floxed (f) in Cre-negative mice (Cre−), while a 1.2-kilobase band shows NEMO deletion in Cre-positive (Cre+) mouse liver. (B) Semiquantitative reverse-transcription PCR from liver RNA using specific primers for GAPDH and NEMO. (C) Absence of NEMO protein was evidenced by Western blot analysis with 30 μg of whole liver extracts using antibodies against NEMO and β-actin. (D) Immunohistochemistry on frozen liver sections from both NEMOf/f and NEMOΔLPC mice was performed using antibody against NEMO. (E) IKK1-IKK2 complex formation was assessed by immunoprecipitation (ip) with an IKK2 and further Western blot with an IKK1 antibody. Gastroenterology 2007 132, 2504-2517DOI: (10.1053/j.gastro.2007.03.045) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 TNF causes liver damage and liver apoptosis in NEMOΔLPC mice, which exhibit lack of NF-κB activation and its dependent gene transcription. Liver damage after intravenous injection of 6 μg/kg of recombinant TNF was evaluated by (A) alanine aminotransferase (ALT) and AST serum levels at 3, 6, 9, and 12 hours after TNF injection. Values are mean ± SD (n = 4). **P < .01 (NEMOΔLPC vs NEMOf/f). Apoptosis was evaluated by (B) TUNEL assay on frozen liver sections and (C) caspase-3 activity assay on liver extracts at 3, 6, and 12 hours after TNF. This result is expressed in fluorescence arbitrary subunits. Values are mean ± SD (n = 4). *P < .05 and **P < .01 (NEMOΔLPC vs NEMOf/f). (D) A total of 5 μg of nuclear proteins from NEMOf/f and NEMOΔLPC mice 30 and 60 minutes after TNF was subjected to an electrophoretic mobility shift assay with a consensus NF-κB probe. Supershift was performed with anti-p50 and anti-p65 antibodies using protein samples from NEMOf/f 60 minutes after stimulus. Western blot analysis of 30 μg of whole cell liver extracts using (E) IκB-α and (F) Bcl-2 antibodies. GAPDH was used as loading control. All samples shown are representative of 4 animals per time point and genotype. Gastroenterology 2007 132, 2504-2517DOI: (10.1053/j.gastro.2007.03.045) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 TNF causes apoptosis of primary hepatocytes from NEMOΔLPC mice due to their complete inability to activate NF-κB. (A) TUNEL assays of hepatocytes isolated from NEMOΔLPC mice showed some basal apoptosis and a strong increase in the number of apoptotic cells 3 hours after TNF stimulation (30 ng/mL), while cells derived from NEMOf/f mice remained unaffected. (B) Quantification of apoptosis in percentage of TUNEL-positive cells relative to number of DAPI nuclei per power field (original magnification 200×). **P<.01 (NEMOΔLP vs NEMOf/f). (C) Immunocytochemistry using a p65 antibody detected by secondary antibody conjugated with Cy3 (red labeling) showed nuclear colocalization with DAPI staining (blue) 30 and 60 minutes after TNF stimulation demonstrating NF-κB translocation in hepatocytes from NEMOf/f mice, which was not detected when NEMOΔLPC hepatocytes were applied. Gastroenterology 2007 132, 2504-2517DOI: (10.1053/j.gastro.2007.03.045) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 I/R causes death of NEMOΔLPC mice, which evidenced prominent necrosis and inflammation. (A) Only 50% of NEMOΔLPC mice survive 6 hours after I/R, and all die within 24 hours. All wild-type mice survived surgery. (B) Serum transaminase levels were measured at 3 and 6 hours after reperfusion. Values are mean ± SD (n = 4). **P < .01. (C) Evaluation of liver damage on tissue sections stained with H&E evidenced necrotic areas (n) and presence of inflammatory cells infiltrated indicated with arrows. Results are representative of liver samples from 4 mice per time point (original magnification 20×). (D) Quantification of polymorphonuclear cells (PMN) was performed on H&E-stained liver sections (power field of 400×) at 0, 3, and 6 hours after reperfusion. Values are mean ± SD (n = 4). *P < .05 (NEMOΔLPC vs NEMOf/f). Gastroenterology 2007 132, 2504-2517DOI: (10.1053/j.gastro.2007.03.045) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Reperfusion of the liver after 60 minutes of ischemia induces higher TNF expression and NF-κB activation in NPCs accompanied by JNK activation and misregulation of IL-6 and STAT-3 signaling in NEMOΔLPC mice. (A) Quantification of liver messenger RNA and serum protein levels by real-time PCR and ELISA from NEMOΔLPC mice and wild-type littermates. Each real-time reaction and ELISA were performed in triplicate. Values are mean ± SD (n = 4). *P < .05 (NEMOΔLPC vs NEMOf/f). (B) NF-κB activation was assessed by immunohistochemistry using a p65 antibody in frozen liver sections from NEMOΔLPC and NEMOf/f mice at 6 hours after reperfusion. Results are representative of liver samples from 4 mice per time point (original magnification 400×). (C) Western blots of 30 μg of whole protein extracts were performed with phospho-JNK and JNK antibodies, respectively. (D) IL-6 gene expression was determined by quantitative real-time reverse-transcription PCR from liver messenger RNA, and protein expression was assessed by ELISA on serum samples. Values are mean ± SD (n = 4). *P < .05 (NEMOΔLPC vs NEMOf/f). (E) STAT-3 activation was observed by protein analysis by Western blot from whole cell liver extracts, while total STAT3 protein expression remained unchanged. Samples shown are representative of 4 animals per time point and genotype. Gastroenterology 2007 132, 2504-2517DOI: (10.1053/j.gastro.2007.03.045) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 NEMOΔLPC mice exhibited earlier and more intense iNOS expression than wild-type littermates along with down-regulation of antioxidant enzymes expression after reperfusion. Immunohistochemistry on liver cryosections using (A) an iNOS and (B) a 3-NT antibody. Results are representative of liver samples from 4 mice per time point (original magnification 160×). Gastroenterology 2007 132, 2504-2517DOI: (10.1053/j.gastro.2007.03.045) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 I/R causes apoptotic cell death in NEMOΔLPC mice. Apoptosis was assessed by (A) TUNEL assay on frozen liver sections from NEMOΔLPC mice and wild-type animals. Results shown are representative of stainings on liver samples from 4 mice per time point. From left to right, TUNEL, DAPI, and merged stainings are shown. (B) Quantification of apoptotic bodies per power field (original magnification 400×) on liver slides stained with H&E. Values are mean ± SD (n = 4). *P < .05 (NEMOΔLPC and NEMOf/f). (C) Caspase-3 enzymatic activity was determined in whole cell liver extracts from NEMOΔLPC and NEMOf/f mice 0, 3, and 6 hours after reperfusion. This result was corrected with the total protein concentration and is expressed in fluorescence arbitrary subunits. Values are mean ± SD (n = 4). *P < .05 and **P < .01 (NEMOΔLPC and NEMOf/f). (D) Western blot analysis in 30 μg of whole cell liver extracts from mice 0, 3, and 6 hours after reperfusion was performed using a Bcl-2 antibody and GAPDH as loading control. Gastroenterology 2007 132, 2504-2517DOI: (10.1053/j.gastro.2007.03.045) Copyright © 2007 AGA Institute Terms and Conditions

Figure 8 Activation of NF-κB in Kupffer cells during the ischemic phase induces a faster and more intense proinflammatory response, oxidative stress metabolites, and adherent molecule expression in NEMOΔLPC mice, sensitizing the liver to reperfusion-induced injury. Immunohistochemistry using (A) p65 and (B) TNF antibodies on liver cryosections from NEMOΔLPC and wild-type littermates 20 minutes after ischemia (original magnification 400×). (C) Western blot analysis of protein extracts from livers of mice 20 and 60 minutes after ischemia using a JNK antibody. Results are representative of liver samples from 4 mice per time point. Immunohistochemistry using (D) iNOS and (E) ICAM-1 antibodies on liver cryosections from NEMOΔLPC and wild-type mice at 60 minutes after ischemia (original magnification 160×). All results are representative of cryosections from 4 mice per time point. Gastroenterology 2007 132, 2504-2517DOI: (10.1053/j.gastro.2007.03.045) Copyright © 2007 AGA Institute Terms and Conditions