Supplementary Online Material

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1 Material and Methods Supplementary Online Material Reagents and antibodies Wortmannin, JNK inhibitor II (Anthra[1,9-cd]pyrazol-6(2H)-one 1,9-pyrazoloanthrone), SB 2358, and PD 9859 were purchased from Calbiochem (San Diego, CA). Benzyloxycarbonyl-Valyl-Alanyl-Aspartyl- (O-methyl)-fluoromethylketone (zvad), benzyloxycarbonyl-phenyl- Alanyl-fluoromethylketone (zfa), benzyloxycarbonyl-leucyl-glutamyl- Histidyl-Aspartyl-fluoromethylketone (LEHD), benzyloxycarbonyl- Aspartyl-(O-methyl)-Glutamyl-(O-methyl)-Valyl-Aspartyl-(O-methyl)- fluoromethylketone (DEVD), and benzyloxycarbonyl-alanyl-alanyl- Aspartyl-(O-methyl)-chloromethylketone (zaad) were purchased from Enzyme Systems Products (Livermore, CA). Cycloheximide, 3- methyladenine, and Phorbol myristate acetate were from Sigma (St Louis). Antibodies to mouse caspase-8, RIP, and Beclin-1 were purchased from Pharmingen (San Diego, CA). Antibodies to phospho-jnk, MKK7, and c- Jun were from Cell Signaling Technology (Beverly, MA). The Atg7 antibody was a kind gift from Dr. William Dunn. Preparation of sirna Non-specific RNAi oligoribonucleotides and RNAi oligoribonucleotides corresponding to the following cdna sequences were purchased from Dharmacon (Boulder, CO): Mouse sequences: CAGTTTGGCACAATCAATA for beclin 1. GTTTGTAGCCTCAAGTGTT for mouse ATG7. CCACTAGTCTGACTGATGA for RIP. TGAGATACTCGAGGTGGAT for MKK7. CATTCGATCTCATTCAGTA for c-jun. GATCGAGGATTATGAAAGA for caspase-8. CAAGGAGUGGUGUUGUUAA for caspase-1. CUUGUCUCUGCUCUUAUGA for caspase-2. UUAGCAAGAUUUGGCGAUA for caspase-3. GACGUUGACUGGCUUGUUC for caspase-9. UGACACGCUAUUUCUACCU for caspase-12. Human sequences: CAGTTTGGCACAATCAATA for beclin 1. GGAGUCACAGCUCUUCCUU for human ATG7.

2 Transfection of sirna.5 nmol RNAi were transfected by Amaxa nucleofection TM, using V solution, program T-2 (Gaithersburg, MD). Cells were then cultured in growth medium for 96 hrs before further analysis. Tissue Culture The mouse L929 cell line and human cell line U937 were obtained from the American Type Culture Collection (Rockville, MD). Mouse RAW264.7 cells were a kind gift from Dr. Richard Siegel. L929 cells were cultured in Dulbecco s modified Eagle s medium with 4.5 g/l glucose. U937, RAW264.7 macrophage cells and mouse peritoneal macrophages prepared by thioglycollate injection were cultured in RPMI 164 medium. Media were supplemented with 2 mm L-glutamine, 1% penicillin/streptomycin solution, and 1% fetal bovine serum (FBS). Cell Death Analysis Cell viability was determined after treatments by staining with propidium iodide (2 µg/ml) and flow cytometric analysis on a FACScan. Percent cell death was quantitated as previously described (26). Detection of the phosphorylated JNK/SAPK: L929 cells were treated with DMSO or zvad for 24 hrs in the presence of 2% FBS. The cell lysate was spun for 1 minutes at 13 rpm. 2 ul of the c-jun beads (SAPK/JNK assay kit from Cell Signaling) were added to the supernatant and incubated overnight at 4 C. Beads were washed 4 times with lysis buffer and resuspended in 5 ul of 1X sample buffer. Samples were boiled for 5 minutes and analyzed by SDS-PAGE and Western blot for phosphorylated SAP/JNK by probing with phospho-jnk antibody. Electron microscopy analyses. Cells were fixed in 3% glutaraldehyde in.1 M MOPS buffer (ph 7.) for 8 hrs at room temperature, 3% glutaraldehyde/1% paraformaldehyde in.1 M MOPS buffer (ph 7.) for 16 hours at 4 o C, post-fixed in 1% osmium tetroxide for 1 hour, embedded in Spurr's resin, sectioned, double stained with Uranyl acetate and Lead citrate, and analyzed using a Zeiss EM 1 transmission electron microscope. For each treatment or control group, at least 1 cells from randomly chosen transmission electron microscopy fields were analyzed for quantification of morphological features. Cells with YDFXROHVZHUHVFRUHGDVDXWRSKDJ\SRVLWLYH&HOOVZHUHVWUDWLILHGDV

3 follows: ( YDFXROHVFHOOYDFXROHVFHOO vacuoles/cell), 3 ( YDFXROHVFHOO6FRUHVZHUHUDQNHGDQGFRPSDULVRQV of treatment to control groups were made using the Mann-Whitney U test using the Statview 5..1 program. Statistical analysis of the differences in Fig.1, panels D, E, F, and G and Fig. 2 A, B, D were significant (p<.1). vacuoles / total cytoplasmic area (%) vacuole area / total cytoplasmic area (%) DMSO zvad Fig. S1 Supplemental Figure S1: morphometric analyses of L929 cells treated with DMSO or zvad. NIH Image software was used to compute fractional surface area occupied by autophagic vacuoles

4 ZVAD hrs ZVAD 8 hrs ZVAD 12 hrs Normal cells Autophagic cells(total) Mild autophagy ( cell with 1~ vacuoles) Moderate autophagy ( cell with 2~ vacuoles) Severe Autophagy ( cell with 3 or 1 1 more vacuoles) Apoptotic cell 4 1 Autophagic and apoptotic features 1 1 Lytic/necrotic cell 3 Vacuolated(but not autophagic) 15 Total Table S1 Supplemental Table S1: Time course for zvad-induced autophagy in L929 cells. The fractions of cells with autophagic features based on TEM were quantitated. vacuolated cells (%) DMSO zvad Fig. S2

5 Supplemental Figure S2: Transmission electron microscopy (TEM) of U937 cells treated for 24 hours with DMSO (a) or zvad (b).scale bar in (a, b), 1 um; (c) The fractions of cells with autophagic features based on TEM were quantitated as described in Fig 1 legend. Sample NS RNAi U937 Beclin-1 RNAi Normal U937 ATG7 RNAi Autophagic cells Apoptotic cells Lytic/necrotic cells Vacuolated cells TOTAL Table S2 Supplemental Table S2: Beclin-1 and ATG7 are required for zvad induced autophagy. The fractions of cells with autophagic features based on TEM were quantitated.

6 RAW DMSO 3-MA wortmannin Supplemental Figure S3: zvad induces cell death in RAW264.7, which can be blocked by autophagy inhibitors. RAW264.7 cells were treated with.1 ug/ml Wortmannin (WM) or 1 mm 3-methyladenine (3-MA) for 1 hr and with 1 um zvad or DMSO for 48 hrs, after which cell loss was quantitated by flow cytometry. 6 5 mouse peritoneal macrophages DMSO 3-MA wortmannin Supplemental Figure S4: zvad induce cell death in mouse peritoneal macrophages, which can be blocked by autophagy inhibitors. Mouse

7 peritoneal macrophages cells were treated with.1 ug/ml Wortmannin (WM) or 1 mm 3-methyladenine (3-MA) for 1 hr and with 1 um zvad or DMSO for 24 hrs, after which cell loss were quantitated by flow cytometry DMSO JNK inhibitor p38 inhibitor Erk inhibitor Fig. S5 Supplemental Figure S5: p38 and Erk signaling are not involved in zvad induced L929 cell death. L929 cells were pretreated with 1 ug/ml JNK inhibitor II, 1ug/ml p38 inhibitor SB 2358, or 1 ug/ml Erk inhibitor PD 9859 for 1 hour, and were then treated with 2 um zvad for 4 hours. % cell loss was quantified by flow cytometry as described in the legend to Fig. 1E.

8 DMSO zvad zfa LEHD DEVD zaad Supplemental Figure S6: Induction of L929 cell death by caspases inhibitors. L929 cells were treated with 1ul/ml DMSO, 2uM zvad, 2 um zfa, 2 um LEHD, 2 um DEVD, or 2 um zaad for 4 hours, and % cell loss was quantified by flow cytometry as described in the legend to Fig. 1E NS C-1 C-2 C-3 C-8 C-9 C-12 Fig. S7 Supplemental Figure S7: Induction of L929 cell death by caspase RNAi. L929 cells were transfected with caspase-1, caspase-2, caspase-3, caspase-8, caspase-9, caspase-12 or nonspecific (NS) RNAi. 11 hours after

9 transfection, % cell loss was quantified by flow cytometry as described in the legend to Fig. 1E. Panels below show the abundance of caspases by Western blot or RT-PCR.. RIP cleaved RIP Fig. S8 Supplemental Figure S8: zvad treatment prevents RIP from constitutive caspase-8 cleavage. L929 cells were treated with zvad for 8 hours, and cleavage of RIP was analyzed by western blot using a RIP antibody. The indicated band is the 42 kd proteolytic product that is characteristic of caspase-8 cleavage (1). References 1. Y. Lin, A. Devin, Y. Rodriguez, Z. G. Liu, Genes & Development 13, (1999).