Supplementary Materials and Methods

Primary Hepatocyte Cultures and Treatment

Mouse hepatocytes were isolated by collagenase perfusion as previously described.33 Cell viability was consistently more than 85% regardless of genetic background of mice. Isolated hepatocytes (0.5×106 cells) were plated on 6-well dishes coated with rat collagen typeⅠin Waymouth’s medium containing 10% fetal bovine serum. After 4 hours, the culture was washed with phosphate-buffered saline and changed to serum-free RPMI1640 medium. Cells were cultivated overnight before treatment with various chemicals as indicated in the Figure legends. Chemicals used in cell culture experiments were the proteasome inhibitor MG-132, the JNK inhibitor SP600125, the p38 MAPK inhibitor SB220025, the GSK3 inhibitor BIO, the MEK inhibitor U0126, the translation inhibitor cycloheximide (Sigma, St. Louis, MO), and the CDK inhibitor R-roscovitine (A.G. scientific, San Diego, CA).

Adenoviral Infection and Induction of Apoptosis

Adenoviruses expressing green fluorescent protein (AdGFP) or substitution mutant of IB (S32A, S36A: IB super-repressor) (AdIkB) have been described previously.33 The recombinant replication-deficient adenoviruses expressing Mcl-1 (AdMcl1-WT), substitution mutants of Mcl-1 (S121A, T163A, or S121A/T163A) (AdMcl1-S121A, AdMcl1-T163A, or AdMcl1-S121A/T163A, respectively) (full-length cDNA clones of human Mcl-1 and mutant Mcl-1 are gifts from Dr. H. Ichijo, Tokyo, Japan),29 or Cre recombinase (AdCre) were generated by AdEasyTM adenoviral system (Stratagene, La Jolla, CA). Hepatocytes were infected with adenoviruses 4 hours after isolation at multiplicity of infection (moi) of 10. The culture was washed with phosphate-buffered saline and changed to serum-free RPMI1640 medium with or without adenoviruses. After 2 hours of incubation, the culture media were changed to serum-free RPMI1640 medium, and cells were incubated for additional 16 hours to allow sufficient transgene expression. For the induction of apoptosis, hepatocytes were sensitized towards TNF by AdIkB infection followed by treatment of 30 ng/ml of recombinant mouse TNF- (R&D Systems, Minneapolis, MN).

Caspase-3 Activity Assay and Quantification of Apoptosis

Caspase-3 activity in primary hepatocytes was measured using a caspase-3 assay kit (AnaSpec, San Jose, CA) according to the manufacturer’s instructions. Apoptosis was quantified by double staining cell cultures with Hoechst 33258 (Invitrogen, Carlsbad, CA) and propidium iodide (Sigma) as previously described.33 Cells with the characteristic changes in nuclear morphology including chromatin condensation, margination, and fragmentation by Hoechst 33258 staining were considered apoptotic. Propidium iodide was used as a marker of necrotic cells, and positive cells were excluded from apoptotic cells. Briefly, cells were stained with both dyes (100μM) for 10 minutes and examined under a fluorescent microscope (Olympus Ⅸ72, Tokyo, Japan). Quantification of apoptotic cells was performed by counting at least 1,000 cells, and is expressing apoptotic cells as a percentage of total cells counted.

Western Blot Analysis and Immunoprecipitation

Preparation of whole-cell protein extracts from cells or frozen liver tissues, electrophoresis of whole-cell protein extracts, and subsequent blotting were performed as previously described.33 Membranes were probed with the following antibodies: anti-caspase-3, anti-cleaved-caspase-3, anti-poly (ADP-ribose) polymerase (PARP), anti-Bcl-XL, anti-Bcl-w, anti-phospho-JNK (Cell Signaling, Danvers, MA), anti-Mcl-1, anti-A1, anti-c-Jun, anti--tubulin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse-Mcl-1 (Rockland, Gilbertsville, PA), and anti-JNK (BD Pharmingen, San Diego, CA). Band densitometry was performed using ImageJ software. For detection of Mcl-1 phosphorylation, protein extracts from AdMcl1-WT or AdMcl1-S121A/T163A infected hepatocytes were immunoprecipitated with anti-Mcl-1 antibody and protein-A/G agarose (Santa Cruz). Immunoprecipitates were then subjected to SDS-PAGE, and Western blot analysis was performed using anti-phospho-serine/threonine (BD Transduction Laboratories, Lexington, KY).

JNK Activity Assay

JNK activity was measured in proteins of primary hepatocytes using a JNK assay kit (Cell Signaling) according to the manufacturer’s instructions.

Quantitative Real-Time Polymerase Chain Reaction

Total RNA was extracted from cells or liver tissues using TRIzol reagent (Invitrogen), and cleaned by an RNeasy kit (Qiagen, Valencia, CA). RNA was reverse transcribed using a high capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative real-time polymerase chain reaction (PCR) with the probe-primer sets of Mcl-1 (Mm00725832_s1), Bcl-XL (Mm00437783_m1), and 18S ribosomal RNA (Hs99999901_s1) (Applied Biosystems) was performed using Taqman analysis (ABI Prism 7000 Sequence Detection System, Applied Biosystems). All assays were normalized with 18S RNA as an internal control.

TNF-induced Liver Injury Model

All experiments were performed on age-matched (8-12 weeks) male animals. Mice were pre-treated with 800mg/kg of D-galactosamine (GalN) (Sigma) for 30 minutes and then injected mouse recombinant TNF- (R&D Systems) intraperitoneally in pyrogen-free saline at a concentration of 12 μg/kg. In some experiments, mice were pre-infected with AdGFP, AdMcl1-WT, or AdCre (1×109 plaque-forming units) 2 days before TNF administration by intravenous injection. For determination of the extent of liver injury, serum levels of alanine aminotransferase (ALT) were measured using a commercial kit (Biotron Diagnostic Inc., Hemet, CA).

Histological Analysis

Livers were removed, fixed with10% buffered formalin, embedded in paraffin, and cut into 5-μm-thick sections. All specimens were stained with hematoxylin-eosin (HE) and evaluated under light microscopy. Terminal deoxy-nucleotyidyl transferase-mediated nick end-labeling (TUNEL) assay was performed using an in situ apoptosis detection kit (Chemicon International, Temecula, CA) according to manufacturer’s instructions. Apoptosis was quantified by counting TUNEL-positive cells in 6 randomly selected high power fields (400×magnification).

Statistical Analysis

Results were expressed as mean ±standard deviation from at least three independent experiments. Data between groups were analyzed by two-tailed t test. A P value of less than 0.05 was considered statistically significant.