Cell pellets were re-suspended in 25 l PBS and lysates obtained by snap freezing in liquid nitrogen. the phosphorylation of Bimel. NGF-mediated safety was dependent on phosphatidylinositol-3 kinase (PI3K) signalling since all above apoptotic events, including manifestation and phosphorylation status of Bimel protein, could be reverted from the PI3K inhibitor LY294002. In contrast, NGF experienced no effect on the TG-mediated induction of the unfolded protein response (improved manifestation of Grp78, GADD34, splicing of XBP1 mRNA) or ER stress-associated pro-apoptotic reactions (induction of C/EBP homologous protein [CHOP], induction and processing of caspase-12). These data show that NGF-mediated safety against ER stress-induced apoptosis happens at the level of the mitochondria by regulating induction and activation of Bim and mitochondrial translocation of Bax. Bax, Bak) and anti-apoptotic (Bcl-2, Bcl-xL) users [8]. The multi-domain users of the Bcl-2 family (which contain Bcl-2 homology domains, BH1, BH2 and BH3) take action on intracellular membranes, including ER and mitochondrial membranes, influencing their permeability towards ions and/or proteins. Their best understood function is at the mitochondrial outer membrane, where different family members either promote or inhibit launch of pro-apoptotic factors including cytochrome c [8]. BH3-only members of the family (e.g. Bad, Bim, PUMA, Noxa, Isobutyryl-L-carnitine Bid) regulate the function of the multi-domain Bcl-2 proteins and induce Bax/Bak-mediated cytochrome crelease [8C10]. BH3-only proteins are controlled transcriptionally (e.g. Bim, PUMA) and/or post-translationally (e.g. phosphorylation of Bim or Bad) [9]. Neurotrophins, such as nerve growth element (NGF) take action through tyrosine kinase (Trk) receptors to provide survival and differentiation signals for neuronal cells during development [11]. Deprivation of NGF in sympathetic neurons and differentiated Personal computer12 cells induces apoptosis [12, 13]. In addition, NGF can also guard cells against oxidative stress or toxin-induced apoptosis [14C18]. NGF promotes survival mainly through activation of the TrkA receptor and intracellular kinase pathways, including the phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways [14, 17, 19, 20]. NGF has also been reported to protect against ER stress-induced apoptosis, however, the molecular mechanism is definitely unclear [15, 17]. The aim of this study was to identify the mechanism by which NGF protects Personal computer12 cells against thapsigargin (TG)-induced ER stress. Personal computer12 cells express TrkA receptors and are responsive to NGF [21]. TG inhibits the sarcoplasmic/ER Ca2+-ATPase pump (SERCA) and causes severe ER stress culminating in apoptosis [22]. We examined the induction by TG of the unfolded protein response (UPR) and activation of the apoptotic execution machinery, and investigated the effect of NGF on each of these TG-induced responses in order to determine its mechanism of safety against lethal ER stress. Materials and methods Materials All chemicals were purchased from Sigma unless normally stated. Ac-Asp-Glu-Val-Asp–(4-methyl-coumaryl-7-amide) (DEVD-AMC) was from your Peptide Institute. Rabbit polyclonal antibodies against caspase-3, cas-pase-9, cleaved caspase-7, phospho-Bad (Ser136) and Bax were from Cell Signalling Systems. Mouse monoclonal anti-Bcl-2, rat monoclonal anti-caspase-12 and rabbit polyclonal anti-actin antibodies were from Sigma. Rabbit polyclonal antibodies against Apaf-1, Grp78 and Bim were from StressGen Biotechnologies. Mouse monoclonal anti-Bcl-xL and rabbit polyclonal anti-CHOP antibodies were from Santa Cruz Biotechnology. Mouse monoclonal anti-cytochrome cantibody was from BD Pharmingen. Goat secondary antibodies conjugated to horseradish peroxidase were from Pierce. Rat pheochromocytoma cells (Personal computer12 cells) were from your ECACC. Mouse nerve growth element-2.5S Grade II (NGF) was from Alomone Laboratory. Plasmid create encoding green fuorescent protein (GFP)-tagged Bax (Bax-GFP) was a kind gift from Prof. Jochen Prehn (Division of Physiology Royal College of Cosmetic surgeons, Dublin, Ireland). Culture and treatment of cells PC12 cells were cultured in Dulbecco’s altered Eagle’s medium supplemented with 10% horse serum, 5% foetal calf serum, 50 U/ml penicillin and 50 g/ml streptomycin as previously explained [18]. For experiments, dishes were coated with poly-L-lysine (10 g/ml for 3 hrs to assist adherence of cells) and cells were seeded at 7 105 per cm2 24 hrs prior to treatments. Cells were treated with 1.5 M TG for times indicated. For determining the effect of NGF, 100 ng/ml NGF was added 2 hrs prior to the addition of TG. Pre-treatment with kinase inhibitors was for 1 hr prior to other treatments. Assessment of cell morphology Cells were harvested by gentle trypsinization and 5 104 cells were cyto-centrifuged onto glass slides (using a Shandon Cytospin 3), air-dried and stained using haematoxylin and eosin. Cell morphology was examined using a Zeiss inverse phase microscope. Three fields for each sample and minimum 300 cells/sample from three different experiments were counted. Detection of caspase-3-like activity Caspase-3-like activity (DEVDase activity) was decided fluorometri-cally as previously explained [23]. Cells were harvested by gentle scraping and washed once in ice-cold phosphate-buffered saline (PBS). Cell pellets were re-suspended.Continuous ER stress induces the proapoptotic transcription issue (TF) CHOP (C/EBP homologous protein), and processing of pro-caspase-12 (pro-C12). kinase (PI3K) signalling since all above apoptotic events, including expression and phosphorylation status of Bimel protein, could be reverted by the PI3K inhibitor LY294002. In contrast, NGF experienced no effect on the TG-mediated induction of the unfolded protein response (increased expression of Grp78, GADD34, splicing of XBP1 mRNA) or ER stress-associated pro-apoptotic responses (induction of C/EBP homologous protein [CHOP], induction and processing of caspase-12). These data show that NGF-mediated protection against ER stress-induced apoptosis occurs at the level of the mitochondria by regulating induction and activation of Bim and mitochondrial translocation of Bax. Bax, Bak) and anti-apoptotic (Bcl-2, Bcl-xL) users [8]. The multi-domain users of the Bcl-2 family (which contain Bcl-2 homology domains, BH1, BH2 and BH3) take action on intracellular membranes, including ER and mitochondrial membranes, affecting their permeability towards ions and/or proteins. Their best understood function is at the mitochondrial outer membrane, where different family members either promote or inhibit release of pro-apoptotic factors including cytochrome c [8]. BH3-only members of the family (e.g. Bad, Bim, PUMA, Noxa, Bid) regulate the function of the multi-domain Bcl-2 proteins and induce Bax/Bak-mediated cytochrome crelease [8C10]. BH3-only proteins are regulated transcriptionally (e.g. Bim, PUMA) and/or post-translationally (e.g. phosphorylation of Bim or Bad) [9]. Neurotrophins, such as nerve growth factor (NGF) take action through tyrosine kinase (Trk) receptors to provide survival and differentiation signals for neuronal cells during development [11]. Deprivation of NGF in sympathetic neurons and differentiated PC12 cells induces apoptosis [12, 13]. In addition, NGF can also safeguard cells against oxidative stress or toxin-induced apoptosis [14C18]. NGF promotes survival largely through activation of the TrkA receptor and intracellular kinase pathways, including the phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways [14, 17, 19, 20]. NGF has also been reported to protect against ER stress-induced apoptosis, however, the molecular mechanism is usually unclear [15, 17]. The aim of this study was to identify the mechanism by which NGF protects PC12 cells against thapsigargin (TG)-induced ER stress. PC12 cells express TrkA receptors and are responsive to NGF [21]. TG inhibits the sarcoplasmic/ER Ca2+-ATPase pump (SERCA) and causes severe ER stress culminating in apoptosis [22]. We examined the induction by TG of the unfolded protein response (UPR) and activation of the apoptotic execution machinery, and investigated the effect of NGF on each of these TG-induced responses in order to identify its mechanism of protection against lethal ER stress. Materials and methods Materials All chemicals were purchased from Sigma unless normally stated. Ac-Asp-Glu-Val-Asp–(4-methyl-coumaryl-7-amide) (DEVD-AMC) was from your Peptide Institute. Rabbit polyclonal antibodies against caspase-3, cas-pase-9, cleaved caspase-7, phospho-Bad (Ser136) and Bax were from Cell Signalling Technologies. Mouse monoclonal anti-Bcl-2, rat monoclonal anti-caspase-12 and rabbit polyclonal anti-actin antibodies were from Sigma. Rabbit polyclonal antibodies against Apaf-1, Grp78 and Bim were from StressGen Biotechnologies. Mouse monoclonal anti-Bcl-xL and rabbit polyclonal anti-CHOP antibodies were from Santa Cruz Biotechnology. Mouse monoclonal anti-cytochrome cantibody was from BD Pharmingen. Goat secondary antibodies conjugated to horseradish peroxidase were from Pierce. Rat pheochromocytoma cells (PC12 cells) were from your ECACC. Mouse nerve growth factor-2.5S Grade II (NGF) was from Alomone Laboratory. Plasmid construct encoding green fuorescent protein (GFP)-tagged Bax (Bax-GFP) was a kind gift from Prof. Jochen Prehn (Department of Physiology Royal College of Surgeons, Dublin, Ireland). Culture and treatment of cells PC12 cells were cultured in Dulbecco’s altered Eagle’s medium supplemented with 10% horse serum, 5% foetal calf serum, 50 U/ml penicillin and 50 g/ml streptomycin as previously explained [18]. For experiments, dishes were coated with poly-L-lysine (10 g/ml for 3 hrs to assist adherence of cells) and cells were seeded at 7 105 per cm2 24 hrs prior to treatments. Cells were treated with 1.5 M TG for times indicated. For determining the effect of NGF, 100 ng/ml NGF was added 2 hrs prior to the addition of TG. Pre-treatment with kinase inhibitors was for 1 hr prior to other treatments. Assessment of cell morphology Cells were harvested by gentle trypsinization and 5 104 cells were cyto-centrifuged onto glass slides (using a Shandon Cytospin 3), air-dried and stained using haematoxylin and eosin. Cell morphology was examined using a Zeiss inverse phase microscope. Three fields for each sample and minimum 300 cells/sample from three different experiments were counted. Detection of caspase-3-like activity Caspase-3-like activity (DEVDase activity) was decided fluorometri-cally as previously described [23]. Cells were harvested by gentle scraping and washed once in ice-cold phosphate-buffered saline (PBS). Cell pellets were re-suspended in 25 l.at room temperature in the dark followed by immediate analysis by flow cytometry (FacsCalibur flow cytometer, Beckton Dickinson). caspase-12). These data indicate that NGF-mediated protection against ER stress-induced apoptosis occurs at the level of the mitochondria by regulating induction and activation of Bim and mitochondrial translocation of Bax. Bax, Bak) and anti-apoptotic (Bcl-2, Bcl-xL) members [8]. The multi-domain members of the Bcl-2 family (which contain Bcl-2 homology domains, BH1, BH2 and BH3) act on intracellular membranes, including ER and mitochondrial membranes, affecting their permeability towards ions and/or proteins. Their best understood function is at the mitochondrial outer membrane, where different family members either promote or inhibit release of pro-apoptotic factors including cytochrome c [8]. BH3-only members of the family (e.g. Bad, Bim, PUMA, Noxa, Bid) regulate the function of the multi-domain Bcl-2 proteins and induce Bax/Bak-mediated cytochrome crelease [8C10]. BH3-only proteins are regulated transcriptionally (e.g. Bim, PUMA) and/or post-translationally (e.g. phosphorylation of Bim or Bad) [9]. Neurotrophins, such as nerve growth factor (NGF) act through tyrosine kinase (Trk) receptors to provide survival and differentiation signals for neuronal cells during development [11]. Deprivation of NGF in sympathetic neurons and differentiated PC12 cells induces apoptosis [12, 13]. In addition, NGF can also safeguard cells against oxidative stress or toxin-induced apoptosis [14C18]. NGF promotes survival largely through activation of the TrkA receptor and intracellular kinase pathways, including the phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways [14, 17, 19, 20]. NGF has also been reported to protect against ER stress-induced apoptosis, however, the molecular mechanism is usually unclear [15, 17]. The aim of this study was to identify the mechanism by which NGF protects PC12 cells against thapsigargin (TG)-induced ER stress. PC12 cells express TrkA receptors and are responsive to NGF [21]. TG inhibits the sarcoplasmic/ER Ca2+-ATPase pump (SERCA) and causes severe ER stress culminating in apoptosis [22]. We examined the induction by TG of the unfolded protein response (UPR) and activation of the apoptotic execution machinery, and investigated the effect of NGF on each of these TG-induced responses in order to identify its mechanism of protection against lethal ER stress. Materials and methods Materials All chemicals were purchased from Sigma unless otherwise stated. Ac-Asp-Glu-Val-Asp–(4-methyl-coumaryl-7-amide) (DEVD-AMC) was from the Peptide Institute. Rabbit polyclonal antibodies against caspase-3, cas-pase-9, cleaved caspase-7, phospho-Bad (Ser136) and Bax were from Cell Signalling Technologies. Mouse monoclonal anti-Bcl-2, rat monoclonal anti-caspase-12 and rabbit polyclonal anti-actin antibodies were from Sigma. Rabbit polyclonal antibodies against Apaf-1, Grp78 and Bim were from StressGen Biotechnologies. Mouse monoclonal anti-Bcl-xL and rabbit polyclonal anti-CHOP antibodies were from Santa Cruz Biotechnology. Mouse monoclonal anti-cytochrome cantibody was from BD Pharmingen. Goat secondary antibodies conjugated to horseradish peroxidase were from Pierce. Rat pheochromocytoma cells (PC12 cells) were from the ECACC. Mouse nerve growth factor-2.5S Grade II (NGF) was from Alomone Laboratory. Plasmid construct encoding green fuorescent protein (GFP)-tagged Bax (Bax-GFP) was a kind gift from Prof. Jochen Prehn (Department of Physiology Royal College of Surgeons, Dublin, Ireland). Culture and treatment of cells PC12 cells were cultured in Dulbecco’s altered Eagle’s medium supplemented with 10% horse serum, 5% foetal calf serum, 50 U/ml penicillin and 50 g/ml streptomycin as previously described [18]. For experiments, dishes were coated with poly-L-lysine (10 g/ml for 3 hrs to assist adherence of cells) and cells were seeded at 7 105 per cm2 24 hrs.Exposure of PC12 cells to TG led to dephosphorylation of Bad on Ser136 detectable after 12 hrs (Fig.?(Fig.5A).5A). with siRNA guarded cells against TG-induced apoptosis. NGF delayed the induction and increased the phosphorylation of Bimel. NGF-mediated protection was dependent on phosphatidylinositol-3 kinase (PI3K) signalling since all above apoptotic events, including expression and phosphorylation status of Bimel protein, could be reverted by the PI3K inhibitor LY294002. In contrast, NGF had no effect on the TG-mediated induction of the unfolded protein response (increased expression of Grp78, GADD34, splicing of XBP1 mRNA) or ER stress-associated pro-apoptotic responses (induction of C/EBP homologous protein [CHOP], induction and processing of caspase-12). These data indicate that NGF-mediated protection against ER stress-induced apoptosis occurs at the level of the mitochondria by regulating induction and activation of Bim and mitochondrial translocation of Bax. Bax, Bak) and anti-apoptotic (Bcl-2, Bcl-xL) members [8]. The multi-domain members of the Bcl-2 family (which contain Bcl-2 homology domains, BH1, BH2 and BH3) act on intracellular membranes, including ER and mitochondrial membranes, affecting their permeability towards ions and/or proteins. Their best understood function is at the mitochondrial outer membrane, where different family members either promote or inhibit release of pro-apoptotic factors including cytochrome c [8]. BH3-only members of the family (e.g. Bad, Bim, PUMA, Noxa, Bid) regulate the function of the multi-domain Bcl-2 proteins and induce Bax/Bak-mediated cytochrome crelease [8C10]. BH3-only proteins are regulated transcriptionally (e.g. Bim, PUMA) and/or post-translationally (e.g. phosphorylation of Bim or Bad) [9]. Neurotrophins, such as nerve growth factor (NGF) act through tyrosine kinase (Trk) receptors to provide survival and differentiation signals for neuronal cells during development [11]. Deprivation of NGF in sympathetic neurons and differentiated PC12 cells induces apoptosis [12, 13]. In addition, NGF can also protect cells against oxidative stress or toxin-induced apoptosis [14C18]. NGF promotes survival largely through activation of the TrkA receptor and intracellular kinase pathways, including the phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways [14, 17, 19, 20]. NGF has also been reported to protect against ER stress-induced apoptosis, however, the molecular mechanism is unclear [15, 17]. The aim of this study was to identify the mechanism by which NGF protects PC12 cells against thapsigargin (TG)-induced ER stress. PC12 cells express TrkA receptors and are responsive to NGF [21]. TG inhibits the sarcoplasmic/ER Ca2+-ATPase pump (SERCA) and causes severe ER stress culminating in apoptosis [22]. We examined the induction by TG of the unfolded protein response (UPR) and activation of the apoptotic execution machinery, and investigated the effect of NGF on each of these TG-induced responses in order to identify its mechanism of protection against lethal ER stress. Materials and methods Materials All chemicals were purchased from Sigma unless otherwise stated. Ac-Asp-Glu-Val-Asp–(4-methyl-coumaryl-7-amide) (DEVD-AMC) was from the Peptide Institute. Rabbit polyclonal antibodies against caspase-3, cas-pase-9, cleaved caspase-7, phospho-Bad (Ser136) and Bax were from Cell Signalling Technologies. Mouse monoclonal anti-Bcl-2, rat monoclonal anti-caspase-12 and rabbit polyclonal anti-actin antibodies were from Sigma. Rabbit polyclonal antibodies against Apaf-1, Grp78 and Bim were from StressGen Biotechnologies. Mouse monoclonal anti-Bcl-xL and rabbit polyclonal anti-CHOP antibodies were from Santa Cruz Biotechnology. Mouse monoclonal anti-cytochrome cantibody was from BD Pharmingen. Goat secondary antibodies conjugated to horseradish peroxidase were from Pierce. Rat pheochromocytoma cells (PC12 cells) were from the ECACC. Mouse nerve growth factor-2.5S Grade II (NGF) was from Alomone Laboratory. Plasmid construct encoding green fuorescent protein (GFP)-tagged Bax (Bax-GFP) was a kind gift from Prof. Jochen Prehn (Department of Physiology Royal College of Surgeons, Dublin, Ireland). Culture and treatment of cells PC12 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% horse serum, 5% foetal calf serum, 50 U/ml penicillin and 50 g/ml streptomycin as previously described [18]. For experiments, dishes were coated with poly-L-lysine (10 g/ml for 3 hrs to assist adherence of cells) and cells were seeded at 7 105 per cm2 24 hrs prior to treatments. Cells were treated with 1.5 M TG for times indicated. For determining the effect of NGF, 100 ng/ml NGF was added 2 hrs prior to the addition of TG. Pre-treatment with kinase inhibitors was for 1 hr prior to other treatments. Assessment of cell morphology Cells were harvested by gentle trypsinization and 5 104 cells were cyto-centrifuged onto glass slides (using a Shandon Cytospin 3), air-dried and stained using haematoxylin and eosin. Cell morphology was examined using a Zeiss inverse phase microscope. Three fields for each sample and minimum 300 cells/sample from three different experiments were counted. Detection of caspase-3-like activity Caspase-3-like activity (DEVDase.Notably, LY294002 alone caused a mild induction of Bimel protein, which is probably due to the reduction of basal PI3K/Akt activity and has previously been reported [35, 36]. that NGF-mediated protection against ER stress-induced apoptosis occurs at the level of the mitochondria by regulating induction and activation of Bim and mitochondrial translocation of Bax. Bax, Bak) and anti-apoptotic (Bcl-2, Bcl-xL) members [8]. The multi-domain members of the Bcl-2 family (which contain Bcl-2 homology domains, BH1, BH2 and BH3) act on intracellular membranes, including ER and mitochondrial membranes, affecting their permeability towards ions and/or proteins. Their best understood function is at the mitochondrial outer membrane, where different family members either promote or inhibit release of pro-apoptotic factors including cytochrome c [8]. BH3-only members of the family (e.g. Bad, Bim, PUMA, Noxa, Bid) regulate the function of the multi-domain Bcl-2 proteins and induce Bax/Bak-mediated cytochrome crelease [8C10]. BH3-only proteins are regulated transcriptionally (e.g. Bim, PUMA) and/or post-translationally (e.g. phosphorylation of Bim or Bad) [9]. Neurotrophins, such as nerve growth factor (NGF) act through tyrosine kinase (Trk) receptors to provide survival and differentiation signals for neuronal cells during development [11]. Deprivation of NGF in sympathetic neurons and differentiated Personal computer12 cells induces apoptosis [12, 13]. In addition, NGF can also guard cells against oxidative stress or toxin-induced apoptosis [14C18]. NGF promotes survival mainly through activation of the TrkA receptor and intracellular kinase pathways, including the phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways [14, 17, 19, 20]. NGF has also been reported to protect against ER stress-induced apoptosis, however, the molecular mechanism is definitely unclear [15, 17]. The aim of this study was to identify the mechanism by which NGF protects Personal computer12 cells against thapsigargin (TG)-induced ER stress. Personal computer12 cells express TrkA receptors and are responsive to NGF [21]. TG inhibits the sarcoplasmic/ER Ca2+-ATPase pump (SERCA) and causes severe ER stress culminating in apoptosis [22]. We examined the induction by TG of the unfolded protein response (UPR) and activation of the apoptotic execution machinery, and investigated the effect of NGF on each of these TG-induced responses in order to determine its mechanism of safety against lethal ER stress. Materials and methods Materials All chemicals were purchased from Sigma unless normally stated. Ac-Asp-Glu-Val-Asp–(4-methyl-coumaryl-7-amide) (DEVD-AMC) was from your Peptide Institute. Rabbit polyclonal antibodies against caspase-3, cas-pase-9, cleaved caspase-7, phospho-Bad (Ser136) and Bax were from Cell Signalling Systems. Mouse monoclonal anti-Bcl-2, rat monoclonal anti-caspase-12 and rabbit polyclonal anti-actin antibodies were from Sigma. Rabbit polyclonal antibodies against Apaf-1, Grp78 and Bim were from StressGen Biotechnologies. Mouse monoclonal anti-Bcl-xL and rabbit polyclonal anti-CHOP antibodies were from Santa Cruz Biotechnology. Mouse monoclonal anti-cytochrome cantibody was from BD Pharmingen. Goat secondary antibodies conjugated to horseradish peroxidase were from Pierce. Rat pheochromocytoma cells (Personal computer12 cells) were from your ECACC. Mouse nerve growth element-2.5S Grade II (NGF) was from Alomone Laboratory. Plasmid create encoding green fuorescent protein (GFP)-tagged Bax (Bax-GFP) was a kind gift from Prof. Jochen Prehn (Division of Isobutyryl-L-carnitine Physiology Royal College of Cosmetic surgeons, Dublin, Ireland). Tradition and treatment of cells Personal computer12 cells were cultured in Dulbecco’s revised Eagle’s medium supplemented with 10% horse serum, 5% foetal calf serum, 50 U/ml penicillin and 50 g/ml streptomycin as previously explained Rabbit Polyclonal to ARFGAP3 [18]. For experiments, dishes were coated with poly-L-lysine (10 g/ml for 3 hrs to assist adherence of cells) and cells were seeded at 7 105 per cm2 24 hrs prior to treatments. Cells were treated with 1.5 M TG for times indicated. For determining the effect of NGF, 100 ng/ml NGF was added 2 hrs prior to the addition of TG. Pre-treatment with kinase inhibitors was for 1 hr prior to other treatments. Assessment of cell morphology Cells were harvested by mild trypsinization and 5 104 cells were cyto-centrifuged onto glass slides (using a Shandon Cytospin 3), air-dried and stained using haematoxylin and eosin. Cell morphology was examined using a Zeiss inverse phase microscope. Three fields for each sample and minimum amount 300 Isobutyryl-L-carnitine cells/sample from three different experiments were counted. Detection of caspase-3-like activity Caspase-3-like activity (DEVDase.