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Supporting Materials Materials and Methods Cells and Culture Conditions. Studies were performed in the rat hepatocyte line RALA255-10G cultured as previously described.1 These cells are conditionally immortalized with a mutant SV40 virus that expresses a temperature-sensitive T antigen.2 Cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) supplemented with 4% fetal bovine serum (Gemini, Woodland, CA), and antibiotics (Invitrogen) at the permissive temperature of 33°C. For experiments, these cells were cultured in Dulbecco’s modified Eagle’s medium, 2% fetal bovine serum, antibiotics, and 1 μM dexamethasone at the restrictive temperature of 37°C for 3 days and then placed in serum-free medium containing dexamethasone for 18 h. Under these conditions, T antigen expression is suppressed and the cells are nontransformed and display a differentiated hepatocyte phenotype.2,3 To inhibit macroautophagy, cells were stably infected with lentiviruses expressing shRNAs to atg5, a critical gene in the macroautophagy pathway.4 Studies were performed in control cells infected with lentiviral vector alone (VEC cells) or cells expressing an Atg5 shRNA (siAtg5 cells), which have been previously described.5 Selected investigations employed cells expressing other distinct Atg5 shRNAs: siAtg5 #2 cells5 and siAtg5 #3 cells. siAtg5 #3 cells express the Atg5 shRNA 5′-GATCCCCGGTTATGAGACAAGAAGATTTCAAGAGAAT TTCGTTGATCACCTGACTTTTTC-3′ which decreased Atg5 levels to an equivalent extent as the other two shRNAs (data not shown). To inhibit CMA, a stable infection with a lentivirus expressing the shRNA 5′-GATCCCCGGAGTACTTATTCTAGTGTTTCAAGAGAATTTCGT TGATCACCTGACTTTTTC-3′ to the lysosomal protein LAMP-2A was generated to create siL2A cells. 1 Cells were treated with menadione (Sigma, St. Louis, MO) at the concentrations shown, the caspase inhibitor Q-Val-Asp-OPh (Q-VD-OPh; MP Biomedicals, Aurora, OH) (10 μM), ammonium chloride (20 mM), leupeptin (100 μM) (Fisher, Pittsburgh, PA) and 3-methyladenine (10 mM) (Sigma). Oleic and palmitic acids (Sigma) were conjugated to bovine serum albumin, as previously described.5 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay. The amount of cell death was quantified by MTT assay.6 At 24 h after menadione treatment, the cell culture medium was replaced by an equal volume of a 1 mg/ml MTT solution, pH 7.4, in Dulbecco’s modified Eagle’s medium. After incubation at 37°C for 1 h, the MTT solution was discarded, and 1.5 ml of 1-propanol was added to solubilize the formazan product. The absorbance of this compound was measured at 560 nm in a spectrophotometer. The percentage of cell death was determined by dividing the optical density of the treated group by the optical density for untreated, control cells, multiplying by 100, and subtracting that number from 100. Fluorescence Microscopy. Steady-state levels of apoptotic and necrotic cells were quantified by fluorescence microscopy after costaining with acridine orange and ethidium bromide,7 as previously described.8 Cells with shrunken cytoplasm and condensed or fragmented nuclei as determined by acridine orange staining were considered apoptotic, and necrotic cells were detected by positive staining with ethidium bromide. A minimum of 400 cells per dish were examined, and the numbers of apoptotic and necrotic cells expressed as a percentage of the total number of cells counted. 2 Lucigenin Assay. ROS production was determined by lucigenin chemiluminescence. Cells were exposed to lucigenin (1 mg/ml) in Krebs-Ringer solution. Levels of chemiluminescence were measured in a microplate reader and normalized to cellular protein. Protein Isolation and Western Blotting. Total protein was isolated as described previously.9 Protein concentrations were determined by the Bio-Rad (Hercules, CA) protein assay according to the manufacturer’s instructions. Membranes were exposed to antibodies that recognized phosphorylated and total JNK1 and JNK2, phosphorylated and total c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated and total extracellular signal-regulated kinase (ERK) 1/2, microtubule-associated protein light chain 3 (LC3), Atg7, caspase 3, caspase 7, poly (ADP-ribose) polymerase (PARP) (Cell Signaling, Beverly, MA), Atg5, beclin 1 (Novus, Littleton, CO), LAMP-1 (Hybridoma Bank, University of Iowa) and -actin (Abcam, Cambridge, MA). The antibodies for LAMP-2A and LAMP-2B were kindly provided by Ana Maria Cuervo (Albert Einstein College of Medicine, Bronx, NY).10 Mitochondrial and cytosolic protein fractions were isolated as previously described.11 Western blotting was performed as above with antibodies to cytochrome c (BD Biosciences), Bax (Santa Cruz), Bid (kind gift of Xiao-Ming Yin, University of Pittsburgh, PA)12 and cytochrome oxidase (MitoSciences, Eugene, OR). Adenovirus Preparation and Infection. To inhibit the mitochondrial death pathway, cells were infected with adenoviruses expressing Bcl-2 or Bcl-XL.13,14 c-Jun function was inhibited by the adenovirus Ad5TAM that expresses TAM-67, a dominant negative c-Jun.15 Ad5LacZ which expresses the Escherichia coli -galactosidase gene,16 served as a control for the nonspecific 3 effects of adenoviral infection. Viruses were amplified in 293 cells, purified by banding twice on CsCl gradients as previously described,17 and titered by plaque assay. Infections were performed at an MOI of 20, as previously described.8 ATP Assay. Intracellular ATP concentrations were determined by the Roche Applied Science (Indianapolis, IN) ATP bioluminescence assay kit HS II using the manufacturer's instructions, as previously described.18 Lactate Assay. Lactate levels were assayed in the cell culture medium by commercial kit (BioVision; Mountain View, CA) using the manufacturer's instructions. Lactate production was measured over a 2 h period and normalized to total cellular protein. Pulse-chase Metabolic Labeling. Rates of degradation of long-lived proteins were measured as previously described.18 Cells were labeled with [3H]valine (60 Ci/mmole) for 48 h at 37°C and then extensively washed and placed in medium lacking radiolabeled valine. Aliquots of medium were removed at 4 and 12 h and proteins in the medium precipitated with trichloroacetic acid. The amount of proteolysis was expressed as the percentage of the initial acid-insoluble radioactivity (protein) transformed into acid-soluble radioactivity (amino acids and small peptides). Total radioactivity incorporated into cellular proteins was determined as the amount of acid-precipitable radioactivity in labeled cells after washing. Lysosomal degradation was calculated as the percentage of protein degradation inhibited by ammonium chloride and leupeptin. Lysosomal protein degradation inhibited by 3-methyladenine was attributed to macroautophagy. 4 Statistical Analysis. All numerical results are reported as mean S.E., and are results from a minimum of three independent experiments. Groups were compared by the Student's t-test with statistical significance defined as P<0.05. Calculations were made with Sigma Plot (Jandel Scientific, San Rafael, CA). Supporting References 1. Jones BE, Lo CR, Liu H, Srinivasan A, Streetz K, Valentino KL et al. Hepatocytes sensitized to tumor necrosis factor- cytotoxicity undergo apoptosis through caspasedependent and caspase-independent pathways. J Biol Chem 2000; 275:705-712. 2. Chou JY. Temperature-sensitive adult liver cell line dependent on glucocorticoid for differentiation. Mol Cell Biol 1983; 3:1013-1020. 3. Chou JY, Yeoh GC. Tyrosine aminotransferase gene expression in a temperature-sensitive adult rat liver cell line. Cancer Res 1987; 47:5415-5420. 4. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell 2008; 132:27-42. 5. Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M et al. Autophagy regulates lipid metabolism. Nature 2009; 458:1131-1135. 6. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65:55-63. 7. Duke RC. Methods of analyzing chromatin changes accompanying apoptosis of target cells in killer cell assays. Methods Mol Biol 2004; 282:43-66. 8. Liu H, Lo CR, Jones BE, Pradhan Z, Srinivasan A, Valentino KL et al. Inhibition of c-Myc expression sensitizes hepatocytes to tumor necrosis factor-induced apoptosis and necrosis. J Biol Chem 2000; 275:40155-40162. 9. Wang Y, Schattenberg JM, Rigoli RM, Storz P, Czaja MJ. Hepatocyte resistance to oxidative stress is dependent on protein kinase C-mediated down-regulation of c-Jun/AP-1. J Biol Chem 2004; 279:31089-31097. 10. Bandyopadhyay U, Kaushik S, Varticovski L, Cuervo AM. The chaperone-mediated autophagy receptor organizes in dynamic protein complexes at the lysosomal membrane. Mol Cell Biol 2008; 28:5747-5763. 5 11. Wang Y, Singh R, Lefkowitch JH, Rigoli RM, Czaja MJ. TNF-induced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway. J Biol Chem 2006; 281:15258-15267. 12. Chen X, Ding WX, Ni HM, Gao W, Shi YH, Gambotto AA et al. Bid-independent mitochondrial activation in tumor necrosis factor -induced apoptosis and liver injury. Mol Cell Biol 2007; 27:541-553. 13. Liedtke C, Plumpe J, Kubicka S, Bradham CA, Manns MP, Brenner DA et al. Jun kinase modulates tumor necrosis factor-dependent apoptosis in liver cells. Hepatology 2002; 36:315-325. 14. Shinoura N, Koike H, Furitu T, Hashimoto M, Asai A, Kirino T et al. Adenovirus-mediated transfer of caspase-8 augments cell death in gliomas: implication for gene therapy. Hum Gene Ther 2000; 11:1123-1137. 15. Bradham CA, Hatano E, Brenner DA. Dominant-negative TAK1 induces c-Myc and G0 exit in liver. Am J Physiol Gastrointest Liver Physiol 2001; 281:G1279-G1289. 16. Iimuro Y, Nishiura T, Hellerbrand C, Behrns KE, Schoonhoven R, Grisham JW et al. NFB prevents apoptosis and liver dysfunction during liver regeneration. J Clin Invest 1998; 101:802-811. 17. Xu Y, Bialik S, Jones BE, Iimuro Y, Kitsis RN, Srinivasan A et al. NF-B inactivation converts a hepatocyte cell line TNF- response from proliferation to apoptosis. Am J Physiol 1998; 275:C1058-C1066. 18. Wang Y, Singh R, Massey AC, Kane SS, Kaushik S, Grant T et al. Loss of macroautophagy promotes or prevents fibroblast apoptosis depending on the death stimulus. J Biol Chem 2008; 283:4766-4777. 6