Apoptosis is orchestrated by a family of cysteine proteases known as the caspases. Fourteen mammalian caspases have been identified, three of which (caspase-3, -6, and -7) are thought to coordinate the execution phase of apoptosis by cleaving multiple structural and repair proteins. However, the relative contributions that the “executioner” caspases make to the demolition of the cell remains speculative. Here we have used cell-free extracts immuno-depleted of either caspase-3, -6, or -7 to examine the caspase requirements for apoptosis-associated proteolysis of 14 caspase substrates as well as nuclear condensation, chromatin margination, and DNA fragmentation. We show that caspase-3 is the primary executioner caspase in this system, necessary for cytochromec/dATP-inducible cleavage of fodrin, gelsolin, U1 small nuclear ribonucleoprotein, DNA fragmentation factor 45 (DFF45)/inhibitor of caspase-activated DNase (ICAD), receptor-interacting protein (RIP), X-linked inhibitor of apoptosis protein (X-IAP), signal transducer and activator of transcription-1 (STAT1), topoisomerase I, vimentin, Rb, and lamin B but not for cleavage of poly(ADP-ribose) polymerase (PARP) or lamin A. In addition, caspase-3 was also essential for apoptosis-associated chromatin margination, DNA fragmentation, and nuclear collapse in this system. Surprisingly, although caspase-6 and -7 are considered to be important downstream effector caspases, depletion of either caspase had minimal impact on any of the parameters investigated, calling into question their precise role during the execution phase of apoptosis. Apoptosis is orchestrated by a family of cysteine proteases known as the caspases. Fourteen mammalian caspases have been identified, three of which (caspase-3, -6, and -7) are thought to coordinate the execution phase of apoptosis by cleaving multiple structural and repair proteins. However, the relative contributions that the “executioner” caspases make to the demolition of the cell remains speculative. Here we have used cell-free extracts immuno-depleted of either caspase-3, -6, or -7 to examine the caspase requirements for apoptosis-associated proteolysis of 14 caspase substrates as well as nuclear condensation, chromatin margination, and DNA fragmentation. We show that caspase-3 is the primary executioner caspase in this system, necessary for cytochromec/dATP-inducible cleavage of fodrin, gelsolin, U1 small nuclear ribonucleoprotein, DNA fragmentation factor 45 (DFF45)/inhibitor of caspase-activated DNase (ICAD), receptor-interacting protein (RIP), X-linked inhibitor of apoptosis protein (X-IAP), signal transducer and activator of transcription-1 (STAT1), topoisomerase I, vimentin, Rb, and lamin B but not for cleavage of poly(ADP-ribose) polymerase (PARP) or lamin A. In addition, caspase-3 was also essential for apoptosis-associated chromatin margination, DNA fragmentation, and nuclear collapse in this system. Surprisingly, although caspase-6 and -7 are considered to be important downstream effector caspases, depletion of either caspase had minimal impact on any of the parameters investigated, calling into question their precise role during the execution phase of apoptosis. apoptotic protease-activating factor-1 piperazine-N,N′-bis(2-ethanesulfonic acid) poly(ADP-ribose) polymerase U1 small nuclear ribonucleoprotein receptor-interacting protein signal transducer and activator of transcription-1 X-linked inhibitor of apoptosis protein DNA fragmentation factor 40/45 polyacrylamide gel electrophoresis Apoptosis is coordinated by a family of cysteine proteases, the caspases, which are activated upon receipt of divergent pro-apoptotic stimuli (1Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4114) Google Scholar, 2Salvesen G.S Dixit V.M Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1934) Google Scholar, 3Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6136) Google Scholar, 4Nicholson D.W. Cell Death Differ. 1999; 6: 1028-1042Crossref PubMed Scopus (1297) Google Scholar, 5Wolf B.B. Green D.R. J. Biol. Chem. 1999; 274: 20049-20052Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar, 6Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2438) Google Scholar). Fourteen caspases have been identified in mammals, a subset of which are thought to be directly involved in the regulation of cell death, with the remainder involved in the maturation of pro-inflammatory cytokines (3Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6136) Google Scholar, 4Nicholson D.W. Cell Death Differ. 1999; 6: 1028-1042Crossref PubMed Scopus (1297) Google Scholar, 6Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2438) Google Scholar, 7Slee E.A. Adrain C. Martin S.J. Cell Death Differ. 1999; 6: 1067-1074Crossref PubMed Scopus (386) Google Scholar). Caspases are synthesized as relatively inactive precursors (zymogens) that require proteolytic processing for activation (3Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6136) Google Scholar, 4Nicholson D.W. Cell Death Differ. 1999; 6: 1028-1042Crossref PubMed Scopus (1297) Google Scholar, 6Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2438) Google Scholar, 8Stennicke H.R. Salvesen G.S. Cell Death Differ. 1999; 6: 1054-1059Crossref PubMed Scopus (153) Google Scholar). Caspase zymogens are typically activated at the onset of apoptosis in two basic ways; those at the apex of signaling cascades are aggregated by caspase adaptor molecules, such as Fas-associated protein with death domain (FADD) or Apaf-1,1 which promote caspase autoactivation, whereas those further downstream are directly activated by apical caspases (6Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2438) Google Scholar, 7Slee E.A. Adrain C. Martin S.J. Cell Death Differ. 1999; 6: 1067-1074Crossref PubMed Scopus (386) Google Scholar, 9Green D.R. Cell. 1998; 94: 695-698Abstract Full Text Full Text PDF PubMed Scopus (1096) Google Scholar, 10Kumar S. Cell Death Differ. 1999; 6: 1060-1066Crossref PubMed Scopus (183) Google Scholar, 11Salvesen G.S Dixit V.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10964-10967Crossref PubMed Scopus (768) Google Scholar). Active caspases promote apoptosis in several ways: by activating other destructive enzymes such as DNases, by promoting mitochondrial cytochrome c release via Bcl-2 family proteins such as BID, and by degrading key structural and regulatory proteins within the cell (3Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6136) Google Scholar, 4Nicholson D.W. Cell Death Differ. 1999; 6: 1028-1042Crossref PubMed Scopus (1297) Google Scholar, 7Slee E.A. Adrain C. Martin S.J. Cell Death Differ. 1999; 6: 1067-1074Crossref PubMed Scopus (386) Google Scholar, 12Martin S.J. Green D.R. Cell. 1995; 82: 349-352Abstract Full Text PDF PubMed Scopus (1258) Google Scholar). Thus far, numerous caspase substrates have been identified, and work is still ongoing to link the caspase-mediated proteolysis of particular substrates with phenotypical changes that take place during apoptosis. Caspases implicated in apoptosis have been divided into two functional sub-groups based upon their perceived roles in this process. Upstream or apical caspases are those that are responsible for initiating the caspase cascade by becoming aggregated upon receipt of a pro-apoptotic stimulus. The latter caspases (caspase-2, -8, -9, -10) tend to have long N-terminal regions (prodomains) with motifs (caspase recruitment domains (CARDs), death effector domains (DEDs)) that are also present in molecules such as Fas-associated protein with death domain (FADD), Apaf-1, and RIP-associated Ich-1/CED homologous protein with death domain (RAIDD), which promote their aggregation (6Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2438) Google Scholar, 7Slee E.A. Adrain C. Martin S.J. Cell Death Differ. 1999; 6: 1067-1074Crossref PubMed Scopus (386) Google Scholar, 9Green D.R. Cell. 1998; 94: 695-698Abstract Full Text Full Text PDF PubMed Scopus (1096) Google Scholar, 10Kumar S. Cell Death Differ. 1999; 6: 1060-1066Crossref PubMed Scopus (183) Google Scholar, 11Salvesen G.S Dixit V.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10964-10967Crossref PubMed Scopus (768) Google Scholar). The second group are downstream, or executioner, caspases (caspase-3, -6, -7) that are thought to be responsible for the actual destruction of the cell and tend to have short or absent prodomains. Much progress has been made in dissecting the routes to activation of the apical caspases, particularly in the context of apoptosis triggered by ligands of the tumor necrosis factor/nerve growth factor receptor superfamily (13Ashkenazi A. Dixit V.M. Science. 1998; 281: 1305-1308Crossref PubMed Scopus (5125) Google Scholar). However, in sharp contrast, the contribution that each executioner caspase makes to the execution phase of apoptosis remains speculative. In particular, although caspase-6 and -7 are widely regarded to act as executioner caspases, their role during the execution phase of apoptosis is obscure. Studies using cells from CASP-3 null mice as well as transformed cell lines devoid of caspase-3 have shown that caspase-3 is important for events such as DNA degradation, nuclear condensation, plasma membrane blebbing, and proteolysis of certain caspase substrates (14Jänicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1704) Google Scholar, 15Jänicke R.U. Ng P. Sprengart M.L. Porter A.G. J. Biol. Chem. 1998; 273: 15540-15545Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 16Zheng T.S. Schlosser S.F. Dao T. Hingorani R. Crispe I.N. Boyer J.L. Flavell R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13618-13623Crossref PubMed Scopus (226) Google Scholar, 17Woo M. Hakem R. Soengas M.S. Duncan G.S. Shahinian A. Kagi D. Hakem A. McCurrach M. Khoo W. Kaufman S.A. Senaldi G. Howard T. Lowe S.W. Mak T.W. Genes Dev. 1998; 12: 806-819Crossref PubMed Scopus (760) Google Scholar). However, some or all of the observed defects in caspase-3-deficient cells could be due to a failure to activate downstream caspases in the absence of caspase-3. In general, studies that have linked cleavage of a caspase substrate to a caspase have used of caspases to remains a caspase that is the at a substrate in is the caspase responsible for this proteolytic in the a dissecting the execution phase of we have cell-free extracts devoid of executioner caspases to the impact of their on events that take place during the demolition phase of apoptosis. We used a cell-free based on from in which apoptosis be triggered by the of cytochrome is well that cytochrome promote caspase activation via the J. R. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, P. D. M. 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We show that caspase-3 is the primary executioner necessary for cleavage of the of the substrates the of and lamin as well as DNA fragmentation and nuclear Surprisingly, depletion of caspase-6 or -7 from cell extracts had minimal impact on any of the parameters investigated, calling into question their role during the execution phase of apoptosis. protein and from and from from was from was from and from and I, and by extracts as E.A. B.B. D.W. Green D.R. Martin S.J. J. Cell Biol. 1999; PubMed Scopus Google Scholar, E.A. S.A. Martin S.J. Cell Death Differ. PubMed Scopus Google Scholar). cells by with of a in cell extracts for at to and other extracts used for as was in the that the cells by three of on at for extracts immuno-depleted of caspases as E.A. B.B. D.W. Green D.R. Martin S.J. J. Cell Biol. 1999; PubMed Scopus Google Scholar, E.A. S.A. Martin S.J. Cell Death Differ. PubMed Scopus Google Scholar). of a of protein with of by for at in or was used as a with to cell Cell extracts immuno-depleted at by of by extracts used and not in a of or of cell of and of and to their with cell or cytochrome c and in the extracts for in the of cytochrome c and and as S.J. S. Green D.R. J. 1995; PubMed Scopus Google Scholar). for substrate proteolysis by as E.A. B.B. D.W. Green D.R. Martin S.J. J. Cell Biol. 1999; PubMed Scopus Google Scholar, S.J. P. G.M. Green D.R. J. PubMed Scopus Google Scholar). for DNA fragmentation using gel as S.J. S. Green D.R. J. 1995; PubMed Scopus Google Scholar). In some within the extracts for apoptotic by with in by protein lamin was by of protein protein from of using the and as E.A. B.B. D.W. Green D.R. Martin S.J. J. Cell Biol. 1999; PubMed Scopus Google Scholar). protein lamin was in the cell-free as protein lamin was by by the contributions of executioner caspases -6, and -7) to the execution phase of we used a cell-free based on extracts of cells in which apoptosis be triggered by of cytochrome We have shown that cell extracts a of apoptosis-associated changes caspase proteolysis of multiple caspase nuclear and fragmentation, and DNA cleavage E.A. B.B. D.W. Green D.R. Martin S.J. J. Cell Biol. 1999; PubMed Scopus Google Scholar, E.A. S.A. Martin S.J. Cell Death Differ. PubMed Scopus Google Scholar, S.J. S. Green D.R. J. 1995; PubMed Scopus Google Scholar, S.J. P. G.M. Green D.R. J. PubMed Scopus Google Scholar). cytochrome as a pro-apoptotic we the of caspase activation events that in the cytochrome in extracts E.A. B.B. D.W. Green D.R. Martin S.J. J. Cell Biol. 1999; PubMed Scopus Google Scholar). the impact of of caspases on downstream caspase activation events be in this context -6, or -7 immuno-depleted from cell extracts using to protein a we also the extracts with or using of caspases from the extracts was by In some we also used extracts from cells that are devoid of caspase-3 due to a in that in the of a into the caspase-3 (14Jänicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1704) Google Scholar). E.A. B.B. D.W. Green D.R. Martin S.J. J. Cell Biol. 1999; PubMed Scopus Google depletion of caspase-3 cytochrome activation of and in the extracts and also activation of a of to cytochrome activation of any other caspase of caspase-6 also to activation of any other executioner caspase but the activation of and in the extracts E.A. B.B. D.W. Green D.R. Martin S.J. J. Cell Biol. 1999; PubMed Scopus Google not caspase-3 important role in the caspase cascade in the cytochrome in to role as effector caspase We the impact of caspase-3, -6, or -7 depletion on proteolysis of the caspase substrate proteins gelsolin, and in this S.H. S. Y. Google Scholar, A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S.J. A. Green D.R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, S. T. C. A. J. Science. 1997; PubMed Scopus Google Scholar). in of caspase-6 and -7 from the extracts had on cytochrome of gelsolin, or In contrast, extracts of caspase-3 of proteolysis of the latter with the of of to a was in the absence of this cleavage has been shown to be by R. D. A. P. Biochem. J. PubMed Scopus Google Scholar, Green D.R. S. G. Cell Death Differ. 1998; 12: Scopus Google Scholar). In contrast, the of the cleavage of was in the absence of caspase-3 but not in the absence of either or The that cleavage was by the of either caspase-3, -6, or -7 was and either that is the caspases with to cleavage or that the apical caspase in the cytochrome caspase cascade is responsible for this cleavage was not with to cleavage of gelsolin, or the that and -7 have substrate N.A. T. M. V.M. S. D.W. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, M. R. D.W. N.A. Cell Death Differ. 1999; 6: PubMed Scopus Google and all are activated in extracts E.A. B.B. D.W. Green D.R. Martin S.J. J. Cell Biol. 1999; PubMed Scopus Google Scholar, E.A. S.A. Martin S.J. Cell Death Differ. PubMed Scopus Google Scholar). the of we extracts devoid of all three caspases -6, -7) and with extracts of caspases. B that whereas extracts of caspases proteolysis of as extracts of all three caspases -6, and -7) to proteolysis of this caspase-6 is not activated in the absence of caspase-3 E.A. B.B. D.W. Green D.R. Martin S.J. J. Cell Biol. 1999; PubMed Scopus Google is that caspase-6 to the caspase-3 and -7 at the of proteolysis but not with to proteolysis of gelsolin, or further the contributions of caspase-3, -6, and -7 to the execution phase of we to several caspase substrates C. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Martin S.J. Green D.R. J. PubMed Scopus Google Scholar, R.U. Porter A.G. J. PubMed Scopus Google Scholar, Martin S.J. Green D.R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus (153) Google Scholar, P. S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, Y. A. Y. Genes Dev. 1999; PubMed Scopus Google Scholar, Genes 1999; PubMed Scopus Google Scholar, H.R. Salvesen G.S. J. 1999; PubMed Scopus Google Scholar, G.S. 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Green D.R. Martin S.J. J. Cell Biol. 1999; PubMed Scopus Google Scholar). However, of extracts immuno-depleted of caspase-6 whereas proteolysis of lamin was proteolysis of lamin B was by of caspase-6 from the extracts and that lamin is by caspase-6 during the execution phase of apoptosis but that lamin B is caspase-3 of the changes that during apoptosis the cell J. PubMed Scopus Google Scholar). caspases are thought to to the nuclear substrate proteolysis and other changes that chromatin condensation, of chromatin to the of the nuclear and fragmentation of was of to the contributions of caspase-3, -6, and -7 to the latter all of which are cytochrome in cell-free in depletion of caspase-3 had a impact on the of with apoptotic in to cytochrome In contrast, depletion of caspase-6 or had impact on events and extracts to DNA fragmentation, whereas extracts devoid of caspase-6 or -7 in this The latter are in with that of caspase-activated DNase which in activation of DNase essential role in apoptosis-associated DNA fragmentation C. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, B.B. M. Green D.R. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google M. A. S. 1998; PubMed Scopus Google Scholar, M. S. 1998; PubMed Scopus Google Scholar, S.J. Green D.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, the failure of caspase-6 and -7 depletion to impact on events further that caspase-3 proteolytic cleavage events during the execution phase of apoptosis that caspase-6 or in any Here we have used a cell-free of extracts from cells to events that the release of mitochondrial cytochrome c during apoptosis. extracts immuno-depleted of caspase-3, or all three caspases, we the role by each caspase in the cleavage of a of proteins known to be for proteolysis during apoptosis. We also the contributions made by caspase-3, -6, and -7 to other apoptosis-associated such as chromatin condensation, chromatin margination, and DNA fragmentation. that caspase-6 and -7 relatively or roles during the execution phase of apoptosis. In contrast, caspase-3 to act as for multiple proteolytic that is the primary executioner caspase into question the that caspase-6 and -7 a role in the phase of apoptosis. has been on the that caspase-6 and -7 have short and have to caspase-3. However, is that caspases a role as of caspase activation within the caspase cascade as destructive caspases in the demolition phase of the caspase substrates that are by caspase-6 and -7 during the execution phase of with the of lamin and not have been identified We the that the of the substrate cleavage events that we have by to be are caspase substrates we have However, we not substrates with any particular and also several substrates lamin protein that are to have cleavage motifs that not be to be by caspase-3. that we have not been to examine using the cell-free is that caspase-6 or -7 be responsible for the membrane changes that promote of apoptotic has been shown that cells from CASP-3 null mice are with to T.S. Schlosser S.F. Dao T. Hingorani R. Crispe I.N. Boyer J.L. Flavell R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13618-13623Crossref PubMed Scopus (226) Google Scholar). remains that caspase-6 or a role in promoting of this to the of the plasma as this has been shown to be P. P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar, S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). caspase-3 and -7 substrate in in is that for caspase-3 N.A. T. M. V.M. S. D.W. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the that is in is activated by at the in the as for caspase-3 for the of the substrate cleavage events The we have to be The latter is by using cells was still in cells a of M. Hakem R. Soengas M.S. Duncan G.S. Shahinian A. Kagi D. Hakem A. McCurrach M. Khoo W. Kaufman S.A. Senaldi G. Howard T. Lowe S.W. Mak T.W. Genes Dev. 1998; 12: 806-819Crossref PubMed Scopus (760) Google Scholar, T.S. S. D. P. Flavell R.A. PubMed Scopus Google Scholar). The present also that the of caspases at to the substrate for a caspase has has long been considered to be a substrate of caspase-3 due to the that the latter in that are to substrate in the of the caspase the relative of the caspases, the in the at which caspases the of the caspases relative to and the of caspase proteins as the present in particular cell the of the nuclear is in cell-free the relative of the caspases and their are cell-free are to the be important to using cells from null mice However, are in with studies that have been using cells from CASP-3 null cleavage of several substrates also in the present lamin as well as nuclear and DNA fragmentation to be (14Jänicke R.U. Sprengart M.L. Wati M.R. Porter A.G. J. Biol. Chem. 1998; 273: 9357-9360Abstract Full Text Full Text PDF PubMed Scopus (1704) Google Scholar, 15Jänicke R.U. Ng P. Sprengart M.L. Porter A.G. J. Biol. 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Flavell R.A. PubMed Scopus Google Scholar). that apoptosis-associated cleavage of several caspase substrates lamin cells T.S. S. Flavell R.A. Cell Death Differ. 1999; 6: PubMed Scopus Google that are not of the cell-free the mice is at with the perceived functional the latter and caspase-3 T.S. S. Flavell R.A. Cell Death Differ. 1999; 6: PubMed Scopus Google Scholar). whereas studies using caspase-3-deficient cells could not the that certain events in apoptosis due to a failure to activate caspases downstream of caspase-3 in the cytochrome -8, E.A. B.B. D.W. Green D.R. Martin S.J. J. Cell Biol. 1999; PubMed Scopus Google to the of extracts to caspase-6 in to and as activation of the latter are by caspase-6 in this context E.A. B.B. D.W. Green D.R. Martin S.J. J. Cell Biol. 1999; PubMed Scopus Google The to this is activation in caspase-3 E.A. B.B. D.W. Green D.R. Martin S.J. J. Cell Biol. 1999; PubMed Scopus Google Scholar). made to from the extracts to the latter was for any of the events that in the absence of of the could this A. and S. J. not remains that some of the defects in extracts are due to the failure to activate However, is that as executioner cells from null mice not to any defects in this G. Y. A. S. A. J.L. J. Genes Dev. 1998; 12: PubMed Scopus Google Scholar). In we have that caspase-3 is the executioner caspase and is not with other executioner caspases, such as caspase-6 and We that caspase-6 and -7 roles in apoptosis was The of the mice be in this and to the precise contributions that each make to the cell death work remains to be with to caspase substrate cleavage events with the and plasma membrane that during apoptosis. We Green and for of and for protein