Download Supporting Materials

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.
NFB 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