Cell, Volume 133

Supplemental Data

TNF- Induces Two Distinct

Caspase-8 Activation Pathways Lai Wang, Fenghe Du, and Xiaodong Wang

Supplemental Experimental Procedures

Reagents

Smac mimetic and the biotinylated compound were synthesized as previously described

(Petersen et al., 2007). z-VAD and MG-132 was obtained from Calbiochem. Cycloheximide was

obtained from Sigma. The following antibodies were used for western-blot: caspase-3 (Cell

Signaling, 9662), caspase-8 (Cell Signaling, 9746), caspase-8 (BD Bioscience, 556466), c-FLIP

(Axxora, ALX-804-428), cIAP1 (R&D, AF8181), cIAP2 (BD Biosciences, 552782), CYLD (Axxora,

ALX-210-910), FADD (Stressgen, AAM-212E), Flag (Sigma, A8592), HA (Roche, 12013819001),

IB- (Santa Cruz Biotech, SC-371), Pan cIAP (R&D, MAB3400), PARP (Cell Signaling, 9542),

RIPK1 (BD Biosciences, 610458), RIPK1 (BD Biosciences, 551041), TNFRI (Santa Cruz Biotech,

SC-8436), TRADD (BD Biosciences, 610572), XIAP (BD Biosciences, 610717).

Plasmids and siRNA Oligoes

c-FLIP cDNA was amplified from a Hela cDNA library with primers containing a N-

terminal Flag epitope. It was then cloned into pCI-neo plasmid (Promega). HA-RIP construct was

generated in a similar fashion, but with an N-terminal HA epitope. FADD-DN (amino acid 81-208

of FADD) cDNA was also amplified using Hela cDNA library and subsequently cloned into a

modified pCI-neo plasmid, resulting in a fusion protein containing three Flag epitopes at the N-

terminus. p3xFLAG-CMV-7 mammalian expression plasmids for cIAP1 and cIAP2 as well as their

E3 ligase mutants H588A and H574A, respectively, were kindly provided by Dr. Chunying Du

(Stowers Institute). cIAP1 with a C-terminal Flag tag and its truncation constructs were amplified

with specific primers accordingly and subcloned into pCI-neo plasmid. For RIPK1-shRNA

construct, eight copies of RIPK1 shRNA (sequence as 5-ccactagtctgacggataa-3) were cloned into

pSuperior.puro vector using the protocol as previously described (Zhong et al., 2005). To generate

RIPK1 scramble expression constructs, RIPK1 cDNA with a C-terminal Flag epitope was first

subcloned into a modified pcDNA4/TO vector (Invitrogen) containing neomycin resistant gene. The

shRNA targeting site on RIPK1 was then mutated at six different sites without affecting amino acid

sequence. For kinase-dead version of RIPK1, lysine 45 was further mutated into alanine.

All siRNAs were purchased from Dharmacon. c-FLIP, caspase-8, RIPK1, and TRADD

were ON-TARGETplus siRNA pools of 4 oligoes. FADD was siGENOME siRNA pools of 4

oilgoes. For cIAP1, cIAP2, XIAP, and CYLD, individual oligo was used (cIAP1 target sequence 5-

TTCGTACATTTCTCTCTTA-3; cIAP2 target sequence 5-AATGCAGAGTCATCAATTA-3;

XIAP target sequence 5-CCAGAATGGTCAGTACAAA-3; CYLD target sequence 5-

GAAGGCUUGGAGAUAAUGA-3).

Recombinant Proteins

GST-TNF- bacterial expression plasmid was kindly provided by Dr. Zhijian Chen (UT

Southwestern Medical Center at Dallas) and GST-TNF- protein was generated as previously

described (Ea et al., 2006). GST-cIAP1 and GST-cIAP2 was expressed in E. coli strain BL21 (DE3)

and purified using Glutathione Sepharose 4B beads (Amersham Biosciences).

Cell Culture

Panc-1 pancreatic carcinoma, T98G glioblastoma, U-2OS osteosarcoma, MIA PACA-2

pancreatic carcinoma, and HEK-293T cells were obtained from ATCC and were cultured in DMEM

supplemented with 10% fetal bovine serum (FBS, Sigma) and 100-units/ml penicillin/streptomycin

(Hyclone). H2009 non-small cell lung carcinoma cells were obtained from the laboratory of Dr.

John Minna (UT Southwestern Medical Center at Dallas). Jurkat leukemia cells and RIPK1

deficient Jurkat cells were kindly provided by Dr. Adrian Ting (Mount Sinai School of Medicine,

New York, NY). H2009 cells and Jurkat cells were grown in HyQ RPMI-1640 medium (Hyclone)

supplemented with 10% FBS and 100-units/ml penicillin/streptomycin.

Methylene Blue Staining

For Supplementary Figure 2A, cell survival was accessed by methylene blue staining.

Briefly, cells were treated as indicated in figure legend. After 48 hours, cells were washed twice

with PBS (Invitrogen) and stained with 2% methylene blue (w/v) in 50% ethanol for 15 minutes with

shaking at room temperature (RT). Cells were then washed twice with PBS and air-dried before

being photographed.

Generation of Stable Cell Lines

To generate c-FLIP or FADD-DN overexpression cells, linearized plasmids were transfected

into Panc-1 cells as previously described. Forty-eight hours post transfection, cells were split and

placed in complete medium containing 1 mg/ml G418 (Calbiochem). After 2 to 3 weeks, clones

were lifted and tested for expression of the transgene. To generate H2009 tetracycline repressor

expression cells, an expression plasmid containing tetracycline repressor (TetR) was transfected into

H2009 cells and cells were selected with 10 g/ml blasticidine (Invitrogen). Inducible RIPK1-

shRNA construct was then stably introduced into H2009-TetR cells, selected with 1 g/ml

puromycin (Calbiochem). Finally, inducible wild type or kinase-dead version of RIPK1 rescue cells

were generated by transfecting RIPK1-shRNA stable cells with RIPK1 scramble expression

constructs and selected with 1 mg/ml G418.

Fluorogenic Caspase-3 Activity Assay

Cells were treated and subsequently pelleted. Cell pellet was resuspended in Buffer A (20

mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF,

and Complete Protease Inhibitor; Roche). The resuspended cell pellet was incubated on ice for 20

minutes before cells were broken by freezing in liquid nitrogen followed by thawing in 37C

waterbath repetitively for 3 times. The resulting broken cell mixture was centrifuged at 20,000 g for

30 minutes. Protein concentration of the supernatant was determined by Commassie Plus Protein

Assay Kit (Pierce). Twenty micro-grams of cell lysates were incubated with 10 M fluorogenic

caspase-3 substrate (Calbiochem) in a 20 l system. Fluorescence reading was carried out as

previously described (Nijhawan et al., 2003).

Preparation of Cell Lysates with Lysis Buffer

Cell pellet was resuspended in lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10%

glycerol, 1% Triton X-100, 1 mM Na3VO4, 25 mM -glycerol-phosphate, 0.1 mM PMSF, and

Complete Protease Inhibitor) and vortexed for 20 seconds. The resuspended cell pellet was

incubated on ice for 20 minutes and centrifuged at 20,000 g for 30 minutes. Supernatant was

collected for further analysis.

Supplemental References

Ea, C.K., Deng, L., Xia, Z.P., Pineda, G., and Chen, Z.J. (2006). Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol Cell 22, 245-257. Micheau, O., and Tschopp, J. (2003). Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes. Cell 114, 181-190. Nijhawan, D., Fang, M., Traer, E., Zhong, Q., Gao, W., Du, F., and Wang, X. (2003). Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation. Genes Dev 17, 1475-1486. Zhong, Q., Gao, W., Du, F., and Wang, X. (2005). Mule/ARF-BP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis. Cell 121, 1085-1095.

Supplemental Figures

Figure S1. Smac mimetic induces TNF--dependent cell death in tumor cells from a variety of

tissue origins.

(A) T98G glioblastoma cells, (C) MIA PACA-2 pancreatic carcinoma cells, and (D) U-2OS

osteosarcoma cells were treated for 48 hours. (B) H2009 lung carcinoma cells were treated for 72

hours as indicated. Cell survival was determined by measuring ATP levels. Data were represented

as mean + standard deviation of duplicates. All experiments were repeated at least three times with

similar results.

Figure S2. TNF- plus Smac mimetic induces caspase activation, and subsequent apoptosis.

(A) Panc-1 pancreatic carcinoma cells were treated with 100 nM Smac mimetic, 100 ng/ml TNF-,

and/or 20 M z-VAD for 72 hours. Cell viability was determined by methylene blue staining as

described in Experimental Procedures. (B) Same cell lysates used in Figure 1B were used for

caspase-8, caspase-3, PARP, and -actin western-blot analysis. All experiments were repeated at

least three times with similar results.

Figure S3. Smac mimetic has no effect on TNF--induced NF-B activation.

HEK-293T cells were transfected with a firefly luciferase reporter construct containing

three NF-B binding sites together with a renilla luciferase plasmid as an internal control.

Forty-eight hour post transfection, cells were treated and were harvested after another 24

hours for luciferase activity using Dual-Glo Luciferase Assay System. Fold activation

was calculated by normalizing firefly luciferase activity/renilla luciferase activity of each

treatment to that of control treatment. Data were represented as mean + standard

deviation of duplicates. Experiments were repeated at least three times with similar

results.

Figure S4. c-FLIP cleavage requires caspase-8.

(A) Panc-1 cells were transfected with caspase-8 siRNA or control luciferase (Luc) siRNA. For