Cell Survey www.abcepta.com 2 2 1 1 1 S.Gramatikova PhD, K.Gramatiko PhD, J.Mountzouris PhD, T.Gilliam & C.Wu PhD

(1) Abcepta Inc., 10320 Camino Santa Fe, Ste G, San Diego, CA 92121 (2) Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037 ABGENT has hundreds of /survival-related antibodies which cover key targets for , autophagy, necroptosis, and other programed cell death processes. Visit www.abgent.com for a complete listing. Cell Death

Selected Abgent Products Comparison of different cell death programs 8 NECROPTOSIS Caspase 3

Figure Target Tissue/Cell line Cat # A. B. zVAD inhibitor Apoptosome formation CALCIUM- AIF/PARP- A. BAD Human lung carcinoma AP1314b BID CHARACTERISTICS APOPTOSIS NECROPTOSIS AUTOPHAGIC ONCOSIS MEDIATED DEPENDENT B. PUMA Human breast carcinoma AP1317a Cytochrome c DNA BMF release fragmentation C. CASP1 Human hepatocarcinoma AT1400a Chromatin condensation, Mitochondrial dysfunction, Autophagic Membrane whorls Mild chromatin Cellular D. CASP3 Human lung carcinoma AP7563c BAX Morphology nuclear fragmentation, membrane rupture, ER vacuoles, membrane condensation swelling BCL2 BCL2 AIF release apoptotic bodies swelling, increase of ROS rupture E. DNMT1 Human carcinoma (HeLa) AT1805a C. D. BAX F. CBX5 Human carcinoma (HeLa) AT1411a APOPTOSIS Oxidative stress, death Trophotoxicity, TNF, Serum, Calcium entry, DNA damage, Ischemia, TNFR activation G. CASP6 Mouse liver tissue AP1313b Triggers receptors, viral infections, damage-induced lesions, starvation, CDK5 signaling glutamate, NO excitotoxicity NECROPTOSIS hypoxia, etc. ischemia, antimycin A aggregates deg mutants H. MLL3 Transfected 293T cells AP6184a CypD I. AIF1 Human leukemia cells K562 AT1077a Energy collapse Mediators , BH family, etc. ERK2, NUR77 Atg orthologs Calpains, cathepsins PARP, AIF JNK

J. BIRC5 Transfected 293T cells AT1299a ATP depletion E. F. AIF release CYLD Membrane Caspase inhibitors, Necrostatins, Ca2+ chelators, 3-Methyladenine, Calreticulin, PARP inhibitors JNK inhibitors, K. SPP1 Human carcinoma (HeLa) AT4028a permeabilization TOP1 inhibitors, , PARP inhibitors, U0126, bafilomycin A1, mTOR, calpain inhibitors glycine L. JUN Transfected 293T cells AP1984d Deubiquitination Cathepsins Inhibitors 2+ VEGF, zVAD, NO, etc. DN NUR77, CypD inhibitors JNK inhibitors? RIP1 Ca overload PARP M. PARP1 Transfected 293T cells AP6373a C. elegans Some excito- Examples Type I and nuclear pcd Type III and cytoplasmic pcd Type II pcd Ischemic pcd TLR signaling deg mutants toxic pcd Necrostatins Calpains Liver 293T K562 293T HeLa 293T 293T Type 1 IFN family ROS 250 150 250 95 250 72 541 Table 1. Alternative programed cell death (pcd) processes. Necroptosis is a cellular mechanism of necrotic cell death induced by apoptotic stimuli 55 under conditions where apoptotic and/or autophagic execution are prevented. Abbreviations for Fig. 1 and Table 1: AIF, apoptosis-inducing factor; BAX, BCL2-as- 37 55 130 36 25 Fig. 1. Crosstalk between apoptosis and programed (necropto- sociated X protein; BCL2, B-cell CLL/lymphoma 2; BID, BH3 interacting domain death agonist; BMF, Bcl2 modifying factor; CDK5, cyclin-dependent kinase 5; CYLD, 20 36 sis). Caspase 8-mediated degradation of RIP1 (receptor-interacting protein kinase 1) is a major cylindromatosis (turban tumor syndrome); CypD, cyclophilin D; deg, degenerin; ERK2, mitogen-activated protein kinase 1; IFN, interferon; JNK, mitogen-activated 15 molecular switch between apoptosis and necroptosis. Necroptosis centers on the activation of RIP1. As protein kinase 8; mTOR, mechanistic target of rapamycin; NO, nitric oxide; NUR77, nuclear receptor; DN Nur77, dominant negative Nur77; PARP, poly (ADP- opposed to apoptosis, necroptosis does not engage apoptotic regulators such as caspases, BCL2 family ribose) polymerase; ROS, reactive oxygen species; TLR, Toll-like receptor; TNF, ; TNFR, tumor necrosis factor receptor; TOP1, DNA topoisomerase 17 10 15 55 members, or cytochrome c (1-7). 1; U0126, inhibitor of MEK kinase; zVAD, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone, a caspase inhibitor (1-7).

G. H. I. J. K. L. M.

Protein Associations

Identification Protocol Statistical Distribution Inferred Network Fig. 2 siRNA screening for re- quired in necroptosis. siRNA screen of the Oxidoreductase, 10 mouse genome for regulators of necroptosis identified Extracellular matrix, 10 TNF TTRAP SPATA2* TRAF7 a set of 432 genes that regulate necroptosis, a subset siRNA transfection of L929 Protease, 11 Nucleic acid binding, 65 of 32 genes that are associated with RIP1 kinase, 32 genes required for apoptosis, and 7 genes involved in Transporter, 13 cells targeting 16,873 genes both necroptosis and apoptosis. zVAD, carbobenzoxy- valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone; Cytoskeleton, 14 TRAF5 TRAF6 CYLD* n CX, cycloheximide (7).

re e Hydrolase, 16 Fig. 3 Classification of 432 selected pro- zVAD - induced TRAF3 TRAF2 TRAIP teins from the necroptosis screen into (A)

y s c RIPK1 molecular function and (B) biological process catego-

necroptosis r ries. Genes for which no annotations could be assigned

a Signaling, 21 TRAF1 Receptors, 50 HSP90AA1 IKBKG were excluded from the analysis. Categories with at least ten genes are displayed. The number of genes assigned Viability assay pri m Transferase, 20 to each category are shown in gray (7). CASP3* TNFRSF1A* CASP8* GRB2 Fig. 4 Molecular interactions of TNF- induced necroptotic . A, Crosstalk Regulatory, 24 Transcription, 43 CASP7* CASP10* between necroptosis-related proteins, positively identi- Selection of 666 genes required A fied in the secondary screen, with apoptotic (*) and other signaling molecules. The network was generated by using data curated from the literature. Number for zVAD - induced necroptosis Lipid, fatty acid, steroid metabolism, 13 Apoptosis, 10 KCNIP1 BCL2* BMF PARP2 B, of protein binding partners for necroptotic proteins se- Carbohydrate metabolism, 13 lected from the secondary screen. Proliferation & differentiation, 13 Signaling, 95 Neuronal activities, 13 Caspase Necroptosis-related TNF-signaling associated Other Protein abbreviations: ATP6V1G2, vacu- Cell structure & motility, 18 TNF - induced zVAD - induced TNF+CX - induced A olar ATP synthase subunit G2; CASP, caspase; COM- MD4, COMM domain containing 4; EIF5B, eukaryotic ree n Intracellular protein traffic, 19 necroptosis necroptosis apoptosis translation initiation factor 5B; DEFB1, defensin, beta 1; DPYSL4, dihydropyrimidinase-like 4; JPH3, s

y s c >193 Cell cycle, 18 junctophilin 3; HOXA3, homeobox A3; HSPBAP1,

tne r >43 32 genes 432 genes 32 genes r Molecular interactions heat shock associated protein 1; GALNT5, N-acetyl- Transport, 26 of necroptosis proteins galactosaminyltransferase 5; GRB2, growth factor

Nucleic acid ot ein pa receptor-bound protein 2; KCNIP1, Kv channel inter- metabolism, 80 acting protein 1; MAG, myelin associated glycoprotein; 13 10

seconda r Sensory perception, 27 RAB25, RAB25, member RAS oncogene family; SPA- 7 6 6 5 4 4 2 2 2 1 1 1 1 1 1 1 1 1 TA2, spermatogenesis associated 2; TMEM57, trans-

7 genes positive in: of p r Number membrane protein 57; TNFAIP8L1, tumor necrosis 7 G A2 Immunity & defense, 28 5 SL4 AP1 T XA3 PVR

BMF factor, alpha-induced protein 8-like 1; , M A TNFRSF1A A CYLD JPH3

TNF / zVAD / TNF+CX B ARP2 GRB2 CNIP1 Protein metabolism EIF5B DEFB1 AIP8I1 P RAB25 H O K S P DP Y TXNL4B GALNT5 OMMD4 tumor necrosis factor receptor superfamily, member 1A; TMEM HSP B TP6V1G2 Development, 38 C

& modification, 48 TN F TNFRSF1A B A TRAIP, TRAF interacting protein; TRAF, TNF receptor- associated factor 1; TTRAP, TRAF and TNF receptor as- Fig. 2 Fig. 3 Fig. 4 sociated protein; TXNL4B, thioredoxin-like 4B.

Protein Markers Product Abbreviations TNFα Necroptosis CDK4 APOPTOSIS DNA REPAIR TNFR BAD: BCL2-associated agonist of cell death; BCL-X/BCL-2 binding protein; BCL2-binding component 6; BBC2 Membrane PUMA: BCL2 binding component 3; BBC3 Caspase 1, 3, 6, 7 & 8; p53; DNA ligase III; CASP9 HSP90AA1 CASP1, -3, -6: caspase 1, -3, -6 Granzyme B; PARP3; XRCC1, 5 & 6; PCNA; Death domain RIP1 DNMT1: DNA (cytosine-5-)-methyltransferase 1; CXXC finger protein 9; DNA methyltransferase 1 BCL2; NFκB; CDKN1A Werner syndrome Kinase domain CBX5: chromobox homolog 5 (HP1 alpha homolog, Drosophila); heterochromatin protein 1 homolog alpha

Autophagy Lipoxygenases XIAP MLL3: myeloid/lymphoid or mixed-lineage leukemia 3; histone-lysine N-methyltransferase, H3 lysine-4 specific CASP3 DIABLO AIF1: allograft inflammatory factor 1; interferon gamma responsive transcript; ionized calcium-binding adapter poly (ADP-ribose) JNK activation Phospholipase A polymerase 1 (PARP1) Survivin (BIRC5) BIRC5: baculoviral IAP repeat-containing 5; apoptosis inhibitor 4; survivin variant 3 alpha PARP1 & PARP2 ROS formation & Sphingomyelinase SPP1: secreted phosphoprotein 1; bone sialoprotein I; osteopontin protein partners mitochondrial dysfunction Survivin RASA1 CDCA8 JUN: jun oncogene; Jun activation domain binding protein; v-jun avian sarcoma virus 17 oncogene homolog PARP1: poly (ADP-ribose) polymerase 1; ADP-ribosyltransferase (NAD+; poly (ADP-ribose) polymerase) DNA topoisomerase I (TOP1) AURKB

TRANSCRIPTION CELL CYCLE CASP7 DNA damage or drugs XPO1 References Transcription factors; CENPA & B; MYBL2; INCENP Retinoid receptors; Nucleophosmin 1; TOP1 cleavage complex CDC2 1. Bredesen DE. (2007) Key note lecture: toward a mechanistic taxonomy for cell death programs. Stroke 38(2 Suppl), pp.652- DNA topoisomerase I BUB3 Replication Caspase 660. Transcription Kinase 2. Galluzzi L and Kroemer G. (2008) Necroptosis: a specialized pathway of programmed necrosis. Cell 135(7), pp.1161-1163. Covalent TOP1 complex Fig. 7 Regulatory protein 3. Degterev A and Yuan J. (2008) Expansion and evolution of cell death programmes. Nat Rev Mol Cell Biol. 9(5), pp.378- Fig.6 Signaling events associated with TOP1. DNA damage and drugs induce 390. ATM ATR reversible TOP1 cleavage complexes, subsequently converted to irreversible by replication and tran- BER complex scription. Irreversible TOP1 complexes can also be formed during apoptosis. DNA repair engages 4. Golstein P and Kroemer G. (2007) Cell death by necrosis: towards a molecular definition. Trends Biochem Sci. 32(1), pp.37- 43. PARP1 NFκB multiple signaling events, including the BER (base excision repair) complex and the RAD51/ PARP RAD52-associated homologous recombination. Cell-cycle arrest facilitates DNA repair and involves XRCC1 ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and RAD3 related) kinases, 5. Zong WX and Thompson CB. (2006) Necrotic death as a cell fate. Genes & Dev. 20, pp.1-15. DNA ligase CHK1/2 as well as the checkpoint homologs CHK1 and CHK2. Additional molecules, associated with 6. Beneke S, Bürkle A. (2007) Poly(ADP-ribosyl)ation in mammalian ageing. Nucleic Acids Res. 35(22), pp.7456-7465. DNA polβ checkpoint regulation, include CDC25, histone H2AX, MRN complex, and BRCA1. p53 activates apop- Packed chromatin Relaxed chromatin APEX1 XIAP tosis both directly and by transactivating pro-apoptotic genes. p21 is a p53-inducible , which 7. Degterev A, Hitomi J, Germscheid M, Chʼen IL, Korkina O, Teng X, Abbott D, Cuny GD, Yuan C, Wagner G, Hedrick SM, Gerber DNA repair OFF, Transcription OFF DNA repair ON, Transcription ON inhibits CDKs (cyclin-dependent kinases). TOP1 inhibitors suppress apoptosis by activating nuclear factor NFκB, which induces the expression of anti-apoptotic genes (12). SA, Lugovskoy A and Yuan J. (2008) Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. RAD51 CDC25 p53 4(5), pp.313-321. A Fig.7 Molecular interactions between survivin and its protein part- 8: Sarkar S. (2008) Till death do us part. Med Econ. 85(11), pp.42-46. p21 PI3K ners. The network was generated by using protein-protein interaction data curated from the literature. Fig. 5 Modulation of chromatin structure XRCC2/3 9: Hitomi J, Christofferson DE, Ng A, Yao J, Degterev A, Xavier RJ and Yuan J. (2008) Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell 135(7):1311-1323. by PARP1. A, PARP1 is a chromatin-associated enzyme, RAD52 CDK Protein abbreviations: AKT, v-akt murine thymoma viral oncogene homolog; APEX1, which modifies proteins by poly (ADP-ribosyl)ation, indicated nuclease; AURKB, aurora kinase B; BCL2, B-cell CLL/lymphoma 2; BUB3, budding uninhibited 10. Schreiber V, Dantzer F, Ame JC and de Murcia G. (2006) Poly(ADP-ribose): novel functions for an old molecule. Nat Rev Mol in blue. Poly (ADP-ribosyl)ation is a postranslational modifi- AKT by benzimidazoles 3 homolog; CASP, caspase; CDC, cell division cycle; CDKN1A or p21, Cell Biol. 7(7), pp.517-528. cation in response to DNA strand-breaks. B, Application of cyclin-dependent kinase inhibitor 1A; CENPA, centromere protein A; DIABLO, ; ABGENTʼs PARP1 monoclonal antibody #AT3183a for immu- B DNA repair Cell-cycle arrest Apoptosis INCENP, inner centromere protein antigens; MYBL2, v-myb myeloblastosis viral oncogene ho- 11. Chiarugi A and Moskowitz MA. (2002) Cell biology. PARP-1-- a perpetrator of apoptotic cell death? Science 297(5579), nofluorescent detection of PARP1 in HeLa cells. molog (avian)-like 2; PCNA, proliferating cell nuclear antigen; PI3K, phosphoinositide-3-kinase; pp.200-201. RAD51, RAD51 homolog; XIAP, X-linked ; XPO1, exportin 1; XRCC, Fig. 5 Fig. 6 Kinase Transcription factor Other X-ray repair complementing defective repair in Chinese hamster cells. 12. Pommier Y. (2006) Topoisomerase I inhibitors: camptothecins and beyond. Nat Rev Cancer 6(10), pp.789-802. 13. Altieri DC. (2008) Survivin, cancer networks and pathway-directed drug discovery. Nat Rev Cancer 8(1), pp.61-70.