1. Genetic Inactivation of RelA/p65 Sensitizes Adult Mouse Hepatocytes to TNF-induced Apoptosis In Vivo and In Vitro 
Fabian Geisler, Hana Algül, Stephan Paxian, Roland M. Schmid 
Gastroenterology 
Volume 132, Issue 7, Pages (June 2007)
DOI: /j.gastro
Copyright © 2007 AGA Institute Terms and Conditions

2. Figure 1
Disruption of RelA/p65 in the mouse liver. (A) Inducible deletion of RelA/p65 in relaF/FMxCre mice 2 days after poly(I)-poly(C) treatment occurs predominantly in the liver and lymphoid tissues as assessed by Southern blot analysis. (B) Deletion of RelA/p65 results in the generation of a truncated protein (p65Δ) lacking the Rel Homology Domain and a nuclear localization sequence. Lysates from primary hepatocytes isolated from relaF/F and relaF/F MxCre mice before and 7 days after poly(l)-poly(C) treatment were prepared and subjected to immunoblot analysis with antibodies against RelA/p65 and β-actin as loading control. (C) NF-κB activation in response to TNF is abrogated in primary hepatocytes after induced deletion of RelA/p65 in relaF/FMxCre mice. Protein extracts (10 μg) from primary hepatocytes from relaF/F and relaF/FMxCre mice were stimulated with mTNF (20 ng/mL) for the indicated times and subjected to a gel-retardation assay with an NF-κB consensus probe. In lanes 13/14, antibody for RelA/p65 was added as indicated as supershift control. (D) Hepatocyte-specific deletion of RelA/p65 in relaF/FAlbCre mice. Extracts from primary hepatocytes isolated from 8-week-old relaF/F and relaF/F AlbCre animals were prepared and immunoblot analysis was performed with anti-p65 and β-actin antibodies. (E) Loss of NF-κB activation in response to mTNF (20 ng/mL) in primary hepatocytes isolated from relaF/FAlbCre mice. Gel-retardation assay was performed with an NF-κB consensus probe as indicated in the Figure.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
Deletion of a functional RelA/p65 in hepatocytes sensitizes mice to TNF-induced liver injury. (A) relaF/F, relaF/FMxCre, and relaF/FAlbCre mice were injected IV with various recombinant TNF-preparations (mouse-TNF, mTNF or human-TNF, hTNF, each 12 μg/kg) as indicated. Liver injury was assessed by determination of ALT serum levels before and 6 hours after TNF treatment. Values are mean ± SD for independent animals (n = 3–6). All relaF/FMxCre and relaF/FAlbCre mice died within 12 hours regardless of the TNF preparation used. (B) H&E and TUNEL staining of livers obtained 6 hours after TNF treatment from mice of indicated genetic background. Results shown are representative of those obtained in 4 mice (left panel, original magnification, ×400). In a separate experiment, TUNEL data were quantified at 4 hours after TNF treatment as indicated and expressed as TUNEL-positive cells per ×400 high-power field (right panel). Values are mean ± SD for independent animals (n = 3 for each group). (C) relaF/FMxCre and relaF/FAlbCre mice were injected IP with LPS (2 mg/kg). ALT serum levels (left panel) and TNF serum levels (right panel) were determined after 6 hours and 1 hour, respectively (n = 4). H&E and TUNEL staining of livers obtained 6 hours after LPS treatment from mice of indicated genetic background. Results shown are representative of those obtained in 4 mice (middle panel, original magnification, ×400).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
Induced disruption of RelA/p65 in primary hepatocytes from relaF/FMxCre mice renders these cells highly sensitive to TNF-induced apoptosis. (A) Cell death in primary hepatocytes from relaF/F and relaF/FMxCre animals was determined by measuring LDH release 20 hours after various treatment regimens as indicated. D-Gal (5 mmol/L) and zVAD-fmk (50 μmol/L) were preincubated for 30 minutes before the addition of mTNF. LDH release after lysis of cells in Triton 2% served as maximum control (100% LDH release). (B) Caspase-3 activity in primary hepatocytes from relaF/F and relaF/FMxCre animals 6 hours after various doses of mTNF as indicated in the Figure. zVAD-fmk (50 μmol/L) was added 30 minutes before TNF treatment. Caspase-3 activity is expressed as relative fluorescence units (RFU). (C) Lysates from primary hepatocytes from relaF/F and relaF/FMxCre animals after mTNF stimulation (20 ng/mL) for the indicated times were subjected to immunoblot analysis with antibodies against cleaved PARP and cleaved caspase-3. A nonspecific band (n.s.) served as loading control. (D) Hoechst staining (left panel) for determination of nuclear morphologic alterations 6 hours after mTNF treatment (20 ng/mL) in primary hepatocytes from relaF/F and relaF/FMxCre animals (original magnification, ×400). Apoptosis in Hoechst stained hepatocytes was quantified (right panel). For inducible deletion of RelA/p65 in primary hepatocytes from relaF/FMxCre animals, mice were treated with poly(I)-poly(C) 7 days before the isolation procedure. In all experiments, effective deletion of RelA/p65 was confirmed by Western blot analysis with anti-p65 antibody. Data in A, B, and D represent mean ± SD for independent experiments (n = 3) performed in triplicate wells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
Sensitivity to TNF-induced apoptosis of primary hepatocytes from relaF/FAlbCre and IkkβF/FAlbCre animals correlates with loss of NF-κB binding activity. (A) Effective deletion of IKKβ in hepatocytes from IkkβF/FAlbCre animals. Protein lysates from primary hepatocytes isolated from IkkβF/F and IkkβF/FAlbCre animals were subjected to immunoblot analysis using anti-IKKβ antibody and anti-β-actin antibody as loading control. (B) Viability of hepatocytes isolated from IkkβF/FAlbCre and relaF/FAlbCre animals 16 hours after treatment with mTNF (20 ng/mL) was determined by LDH release. Values are mean ± SD for independent experiments (n = 2) performed in triplicate wells. (C) Lysates from mTNF-treated (20 ng/mL) hepatocytes isolated from IkkβF/FAlbCre and relaF/FAlbCre animals were subjected to immunoblot analysis with antibodies against cleaved PARP and β-actin as loading control. (D) Hoechst staining of primary hepatocytes from IkkβF/FAlbCre and relaF/FAlbCre animals for determination of nuclear morphologic alterations after mTNF-treatment (20 ng/mL) for the indicated time points (original magnification, ×400). (E) Protein extracts (10 μg) from mTNF (20 ng/mL) stimulated hepatocyte cultures isolated from mice with indicated genetic background were subjected to a gel-retardation assay using an NF-κB consensus probe. (F) Sole stimulation of TNFR1 is sufficient to induce rapid apoptosis in RelA/p65-deficient hepatocytes. Primary hepatocytes from relaF/FAlbCre animals were treated with 20 ng/mL mTNF or hTNF for the indicated time points, and lysates were subjected to Western blot analysis with antibodies against cleaved PARP and β-actin as loading control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
TNF-induced apoptosis in RelA/p65-deficient hepatocytes is critically dependent on caspase-8. (A) Untreated, mock-transfected, and caspase-8-RNAi transfected hepatocytes from relaF/FAlbCre mice were treated with mTNF (20 ng/mL) for the indicated times, and respective lysates were subjected to immunoblot analysis with antibodies against cleaved PARP and β-actin as loading control (lower panel). Efficient knockdown of caspase-8 is shown in the upper panel; a nonspecific band served as loading control. Representative results from 2 independent experiments are shown. (B) Caspase-3 activation in mock-transfected and caspase-8-RNAi-transfected hepatocytes from relaF/FAlbCre mice was analyzed after treatment with mTNF (20 ng/mL) for 8 hours. Caspase-3 activity is expressed as x-fold induction vs untreated cells (left panel). Viability was determined by measuring LDH release in supernatants of mock- and caspase-8-RNAi-transfected hepatocytes 20 hours after mTNF (20 ng/mL) treatment. Data shown are mean ± SD obtained from 2 independent experiments performed in triplicate. Asterisk marks statistical significance (P < .05) compared with TNF-stimulated mock-transfected cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
The JNK-pathway in TNF-induced apoptosis in RelA/p65-deficient hepatocytes. (A) TNF-induced JNK activation is prolonged in RelA/p65-deficient hepatocytes. Hepatocyte cultures from relaF/F and relaF/FAlbCre animals were treated for the indicated times with mTNF (20 ng/mL), and lysates were either subjected to immunoblot analysis using anti-phospho-JNK or anti-total-JNK antibodies, or lysates were used for JNK-kinase assay with immobilized c-Jun fusion protein linked to agarose beads as described in the Materials and Methods section. (B) The JNK-inhibitor SP prevents TNF-induced apoptosis in RelA/p65-deficient hepatocytes. Hepatocytes were isolated from relaF/FAlbCre animals and treated with mTNF (20 ng/mL) with or without 60-minute preincubation of cells with the JNK-inhibitor SP (20 μmol/L) or with solvent (DMSO, 0.1%). Hoechst staining was performed after 8 hours of mTNF treatment (upper panel, original magnification, ×400). Apoptosis was quantified in Hoechst stained hepatocytes 6 hours after mTNF treatment (lower panel). Values represent mean ± SD obtained from 2 independent experiments performed in triplicate. (C) RelA/p65-deficient hepatocytes from relaF/FAlbCre animals were treated as in B for the indicated times, and lysates were subjected to immunoblot analysis with antibodies against cleaved PARP, c-FLIP, p-JNK, and JNK. Lysates were also subjected to JNK-kinase assay (lower 2 panels).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
The antioxidant BHA limits duration of JNK-activation and prevents apoptosis in RelA/p65-deficient hepatocytes in response to TNF. Hepatocytes were isolated from relaF/FAlbCre animals and treated with mTNF (20 ng/mL) without or with 30-minute preincubation with the antioxidant butylated hydroxyanisole (BHA, 100 μmol/L) as indicated in the Figure. (A) Apoptosis was quantified in Hoechst stained hepatocytes 6 hours after mTNF treatment (right panel, original magnification, ×400). Values represent mean ± SD obtained from 2 independent experiments performed in triplicate. The left picture shows Hoechst stained cultivated hepatocytes 8 hours after mTNF treatment. (B) Lysates from RelA/p65-deficient hepatocytes treated with mTNF (without or with BHA preincubation) for the indicated times were subjected to Western blot analysis or JNK-kinase assay as described in Figure 6.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

9. Figure 8
Levels of c-FLIPL in response to TNF in RelA/p65-deficient hepatocytes decrease secondarily to caspase activation. (A) Hepatocytes from relaF/FAlbCre animals were treated with mTNF (20 ng/mL) without or with 60-minute preincubation with the proteasome inhibitor MG-132 (20 μmol/L) for the indicated times, and lysates were subjected to immunoblot analysis using antibodies against cleaved PARP, c-FLIP, IκBα, and β-actin. (B) Representative phase contrast images of cultivated RelA/p65-deficient hepatocytes 8 hours after treatment with mTNF (20 ng/mL, left image) or with mTNF + MG-132 (20 μmol/L, right image). (C) Hepatocytes from relaF/FAlbCre animals were treated with mTNF (20 ng/mL) for the indicated times either alone or in the presence of the caspase-8 inhibitor zIETD-fmk (10 μmol/L) or the pancaspase inhibitor zVAD-fmk (50 μmol/L). Lysates were subjected to immunoblot analysis with antibodies against c-FLIP, cleaved PARP, p-JNK, and β-actin. (D) Mock- or caspase-8-RNAi-transfected hepatocyte cultures were stimulated with mTNF (20 ng/mL) for the indicated times. Protein lysates were prepared, and Western blot analysis was performed using anti-cleaved PARP, anti-c-FLIP, and anti-β-actin antibodies. Efficient knockdown was confirmed by analyzing caspase-8 expression (left image).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions