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  • 99
    Millipore caspase substrate ac devd amc
    Caspase Substrate Ac Devd Amc, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomol GmbH ac devd amc
    BAX is required for increases in caspase activity. Cultures were switched to K5+S medium, lysed after 4, 8, 12, 24, 48, or 72 h, and cleavage of <t>DEVD-AMC</t> was determined. Control cultures were switched to K25+S medium and treated identically. Data represent mean ± SD for triplicate measurements from one experiment and are representative of two additional independent experiments.
    Ac Devd Amc, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 90/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem ac devd amc
    Cleavage of GCLC and depletion of cellular GSH during apoptotic cell death. HeLa cells were treated with human TNF (30 ng/ml) and cycloheximide (CHX, 10 μg/ml) for the time period indicated. A: GCLC, GCLM, and pro-caspase-3 were analyzed by immunoblotting. Apoptosis was quantified by assessing apoptotic nuclear morphology of DAPI-stained cells. B: Cellular glutathione levels (GSH+GSSG; closed bars Caspase-3-like activities ( open bars ) were determined using the fluorogenic <t>DEVD-AMC</t> substrate. Data presented are from a representative experiment performed at least three times with similar results.
    Ac Devd Amc, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore acetyl devd amc
    Inhibition of the ERK1/2 pathway blocks BDNF-mediated inhibition of caspase-3-like activity. P7 rats received ICV injections of vehicle or U0126 (1 nmol/animal) followed 15 min later by an ICV injection of vehicle or BDNF (5 μg/animal) just before the carotid ligation and exposure to 2.5 hr of hypoxia. Twenty-four hours after H-I, brain tissues from the hippocampus ( A ) and cortex ( B ) contralateral ( right ) and ipsilateral ( left ) to the ligation were dissected and subjected to <t>DEVD-AMC</t> cleavage assay as described in Materials and Methods. Note that there is no activation of caspase-3-like activity in the group treated with U0126 without subsequent H-I ( No HI ). Data represent the mean ± SEM; * p
    Acetyl Devd Amc, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Peptide Institute ac devd amc
    Renal caspase-like activities were determined kinetically in homogenates of tissue obtained after 24 hours ( a and c ) and 2 hours ( b and d ) of renal reperfusion in a fluorogenic substrate assay in which <t>Ac-YVAD-amc</t> (caspase-1–like) ( a and b ) or <t>Ac-DEVD-amc</t> (caspase-3–like) ( c and d ) served as substrates. Note that these data may represent a more generalized form of caspase activation, as outlined in the main text. Data are expressed as the increase in fluorescence as a function of time, normalized against data obtained from the sham-operated group. The groups that indicate t = 2 on the x-axis received the indicated treatment after 2 hours of reperfusion. All other groups were treated at the time of reperfusion. * P
    Ac Devd Amc, supplied by Peptide Institute, used in various techniques. Bioz Stars score: 91/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson ac devd amc
    N. gonorrhoeae inhibits extrinsic apoptosis induced by TRAIL. (A) N. gonorrhoeae inhibits DNA fragmentation in HL-60 induced by TRAIL. Differentiated HL-60 cells were infected with piliated, Opa-expressing FA1090 at an MOI of 9 and then treated with 100 ng/ml TRAIL. DNA fragmentation was evaluated by flow cytometry, and the percentage of cells exhibiting hypodiploid DNA under each condition is indicated. (B) Average percentage of cells exhibiting DNA fragmentation after infection and/or treatment with 100 ng/ml TRAIL. The data are averages for 3 independent experiments. (C) N. gonorrhoeae infection inhibits TRAIL-induced caspase-3 activity in HL-60 cell lysates. HL-60 cells were infected with piliated strain FA1090 at an average MOI of 15 and then treated with 100 ng/ml TRAIL. Caspase-3 activity was measured using <t>Ac-DEVD-AMC.</t> Data are presented as the relative caspase-3 activity compared to that of uninfected, untreated controls and are representative of 6 independent biological replicates.
    Ac Devd Amc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Amresco ac devd amc
    N. gonorrhoeae inhibits extrinsic apoptosis induced by TRAIL. (A) N. gonorrhoeae inhibits DNA fragmentation in HL-60 induced by TRAIL. Differentiated HL-60 cells were infected with piliated, Opa-expressing FA1090 at an MOI of 9 and then treated with 100 ng/ml TRAIL. DNA fragmentation was evaluated by flow cytometry, and the percentage of cells exhibiting hypodiploid DNA under each condition is indicated. (B) Average percentage of cells exhibiting DNA fragmentation after infection and/or treatment with 100 ng/ml TRAIL. The data are averages for 3 independent experiments. (C) N. gonorrhoeae infection inhibits TRAIL-induced caspase-3 activity in HL-60 cell lysates. HL-60 cells were infected with piliated strain FA1090 at an average MOI of 15 and then treated with 100 ng/ml TRAIL. Caspase-3 activity was measured using <t>Ac-DEVD-AMC.</t> Data are presented as the relative caspase-3 activity compared to that of uninfected, untreated controls and are representative of 6 independent biological replicates.
    Ac Devd Amc, supplied by Amresco, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pharmingen ac devd amc
    Nur77 expression inhibits caspase activation induced by TNF stimulation. ( A ) Caspase-3 activity was examined in vector or Nur77 stable transformants in RelA -/- or TRAF2 -/- cells after TNF stimulation. Caspase-3 activity was measured by the appearance of cleaved caspase-3 in Western blot analyses. ( B ) Fluorometric caspase-3 enzymatic activity was measured by using a specific substrate <t>Ac-DEVD-AMC</t> as described in Methods . ( C ) Caspase-8 enzymatic activity was measured by using the substrate Ac-IETD-AFC as described in Methods .
    Ac Devd Amc, supplied by Pharmingen, used in various techniques. Bioz Stars score: 90/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cayman Chemical ac devd amc
    Nur77 expression inhibits caspase activation induced by TNF stimulation. ( A ) Caspase-3 activity was examined in vector or Nur77 stable transformants in RelA -/- or TRAF2 -/- cells after TNF stimulation. Caspase-3 activity was measured by the appearance of cleaved caspase-3 in Western blot analyses. ( B ) Fluorometric caspase-3 enzymatic activity was measured by using a specific substrate <t>Ac-DEVD-AMC</t> as described in Methods . ( C ) Caspase-8 enzymatic activity was measured by using the substrate Ac-IETD-AFC as described in Methods .
    Ac Devd Amc, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega ac devd amc
    Kinetics of cisplatin-induced DEVDase and caspase 9/ LEHDase activities. 224 cells were treated with 20 μM cisplatin for the indicated time periods in the absence or presence of adeno-dnMEKK. Expression of dnMEKK was induced with DOX at 20 h before cisplatin treatment. Empty bars, cisplatin only; filled bars, cisplatin treatment in the presence of dnMEKK. (A) DEVDase activity on the synthetic substrate <t>Ac-DEVD-AMC</t> was assessed twice for each duplicate sample. The inhibitor DEVD-CHO reduced all activity to background in all parallel samples. Results are shown as fold increase in activity in untreated (control [C]) cells. (B) Caspase 9/LEHDase activity on the synthetic substrate Ac-LEHD-AMC. The inhibitor LEHD-CHO reduced all activity to background in all parallel samples. (C) Cell extracts (30 μg/lane) were analyzed by Western blotting of caspase 9. The 35-kDa cleavage fragment represents the active form. β-TUB., tubulin; CISPL, cisplatin.
    Ac Devd Amc, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PeptaNova GmbH ac devd amc
    Kinetics of cisplatin-induced DEVDase and caspase 9/ LEHDase activities. 224 cells were treated with 20 μM cisplatin for the indicated time periods in the absence or presence of adeno-dnMEKK. Expression of dnMEKK was induced with DOX at 20 h before cisplatin treatment. Empty bars, cisplatin only; filled bars, cisplatin treatment in the presence of dnMEKK. (A) DEVDase activity on the synthetic substrate <t>Ac-DEVD-AMC</t> was assessed twice for each duplicate sample. The inhibitor DEVD-CHO reduced all activity to background in all parallel samples. Results are shown as fold increase in activity in untreated (control [C]) cells. (B) Caspase 9/LEHDase activity on the synthetic substrate Ac-LEHD-AMC. The inhibitor LEHD-CHO reduced all activity to background in all parallel samples. (C) Cell extracts (30 μg/lane) were analyzed by Western blotting of caspase 9. The 35-kDa cleavage fragment represents the active form. β-TUB., tubulin; CISPL, cisplatin.
    Ac Devd Amc, supplied by PeptaNova GmbH, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Peptron Inc ac devd amc
    Kinetics of cisplatin-induced DEVDase and caspase 9/ LEHDase activities. 224 cells were treated with 20 μM cisplatin for the indicated time periods in the absence or presence of adeno-dnMEKK. Expression of dnMEKK was induced with DOX at 20 h before cisplatin treatment. Empty bars, cisplatin only; filled bars, cisplatin treatment in the presence of dnMEKK. (A) DEVDase activity on the synthetic substrate <t>Ac-DEVD-AMC</t> was assessed twice for each duplicate sample. The inhibitor DEVD-CHO reduced all activity to background in all parallel samples. Results are shown as fold increase in activity in untreated (control [C]) cells. (B) Caspase 9/LEHDase activity on the synthetic substrate Ac-LEHD-AMC. The inhibitor LEHD-CHO reduced all activity to background in all parallel samples. (C) Cell extracts (30 μg/lane) were analyzed by Western blotting of caspase 9. The 35-kDa cleavage fragment represents the active form. β-TUB., tubulin; CISPL, cisplatin.
    Ac Devd Amc, supplied by Peptron Inc, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA fluorogenic ac devd amc
    Kinetics of cisplatin-induced DEVDase and caspase 9/ LEHDase activities. 224 cells were treated with 20 μM cisplatin for the indicated time periods in the absence or presence of adeno-dnMEKK. Expression of dnMEKK was induced with DOX at 20 h before cisplatin treatment. Empty bars, cisplatin only; filled bars, cisplatin treatment in the presence of dnMEKK. (A) DEVDase activity on the synthetic substrate <t>Ac-DEVD-AMC</t> was assessed twice for each duplicate sample. The inhibitor DEVD-CHO reduced all activity to background in all parallel samples. Results are shown as fold increase in activity in untreated (control [C]) cells. (B) Caspase 9/LEHDase activity on the synthetic substrate Ac-LEHD-AMC. The inhibitor LEHD-CHO reduced all activity to background in all parallel samples. (C) Cell extracts (30 μg/lane) were analyzed by Western blotting of caspase 9. The 35-kDa cleavage fragment represents the active form. β-TUB., tubulin; CISPL, cisplatin.
    Fluorogenic Ac Devd Amc, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore antibodies ac devd amc
    Kinetics of cisplatin-induced DEVDase and caspase 9/ LEHDase activities. 224 cells were treated with 20 μM cisplatin for the indicated time periods in the absence or presence of adeno-dnMEKK. Expression of dnMEKK was induced with DOX at 20 h before cisplatin treatment. Empty bars, cisplatin only; filled bars, cisplatin treatment in the presence of dnMEKK. (A) DEVDase activity on the synthetic substrate <t>Ac-DEVD-AMC</t> was assessed twice for each duplicate sample. The inhibitor DEVD-CHO reduced all activity to background in all parallel samples. Results are shown as fold increase in activity in untreated (control [C]) cells. (B) Caspase 9/LEHDase activity on the synthetic substrate Ac-LEHD-AMC. The inhibitor LEHD-CHO reduced all activity to background in all parallel samples. (C) Cell extracts (30 μg/lane) were analyzed by Western blotting of caspase 9. The 35-kDa cleavage fragment represents the active form. β-TUB., tubulin; CISPL, cisplatin.
    Antibodies Ac Devd Amc, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson ac devd amc protease assay
    PAT-SM6 mediates cytotoxicity to patient MM cells and MM cell lines by induction of apoptosis. A, MM cell lines were incubated with various concentrations of PAT-SM6 or control (unrelated IgM at highest concentration) for 72 h. Cell death was determined by FACS (Propidium iodide/LDS75 double positive cells). B, CD138-purified primary MM cells at primary diagnosis (n = 8, dots) or relapse (n = 3, rectangle) were incubated for 48 h with PAT-SM6 or isotype in media containing IL-6 and 10% FCS. Dead cells were determined by FACS analysis (Propidium iodide/LDS75 double positive cells). As control, PBMCs from healthy volunteers were treated under the same conditions. In contrast to controls PAT-SM6 significantly induced programmed cell death. C, MM1.S cells were incubated with PAT-SM6 or controls (staurophorin as positive control) for 72 h. AnnexinV and 7AAD positive cells were assessed by flow cytometry. Percentage of 7-AAD/Annexin V double positive cells, Isotype: 14%, PAT-SM6∶79% and positive control: 76%. D, MM1.S cells were incubated with PAT-SM6 or controls (staurophorin or isotype) for 6 h and caspase 3 activation was measured using the <t>AC-DEVD-AMC</t> protease assay. E, Bone marrow cells from a MM patient were CD33-purified to obtain myeloid progenitors and stimulated with PAT-SM6 or controls. No specific binding and subsequently no specific cytotoxicity was observed. F, Cells from the same patient as in E were CD138-isolated and incubated with PAT-SM6 (200 µg/mL) for 24 h. Specific PAT-SM6 binding and killing was observed.
    Ac Devd Amc Protease Assay, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega fluorometric substrate ac devd amc
    PAT-SM6 mediates cytotoxicity to patient MM cells and MM cell lines by induction of apoptosis. A, MM cell lines were incubated with various concentrations of PAT-SM6 or control (unrelated IgM at highest concentration) for 72 h. Cell death was determined by FACS (Propidium iodide/LDS75 double positive cells). B, CD138-purified primary MM cells at primary diagnosis (n = 8, dots) or relapse (n = 3, rectangle) were incubated for 48 h with PAT-SM6 or isotype in media containing IL-6 and 10% FCS. Dead cells were determined by FACS analysis (Propidium iodide/LDS75 double positive cells). As control, PBMCs from healthy volunteers were treated under the same conditions. In contrast to controls PAT-SM6 significantly induced programmed cell death. C, MM1.S cells were incubated with PAT-SM6 or controls (staurophorin as positive control) for 72 h. AnnexinV and 7AAD positive cells were assessed by flow cytometry. Percentage of 7-AAD/Annexin V double positive cells, Isotype: 14%, PAT-SM6∶79% and positive control: 76%. D, MM1.S cells were incubated with PAT-SM6 or controls (staurophorin or isotype) for 6 h and caspase 3 activation was measured using the <t>AC-DEVD-AMC</t> protease assay. E, Bone marrow cells from a MM patient were CD33-purified to obtain myeloid progenitors and stimulated with PAT-SM6 or controls. No specific binding and subsequently no specific cytotoxicity was observed. F, Cells from the same patient as in E were CD138-isolated and incubated with PAT-SM6 (200 µg/mL) for 24 h. Specific PAT-SM6 binding and killing was observed.
    Fluorometric Substrate Ac Devd Amc, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomol GmbH fluorophore substrate ac devd amc
    PAT-SM6 mediates cytotoxicity to patient MM cells and MM cell lines by induction of apoptosis. A, MM cell lines were incubated with various concentrations of PAT-SM6 or control (unrelated IgM at highest concentration) for 72 h. Cell death was determined by FACS (Propidium iodide/LDS75 double positive cells). B, CD138-purified primary MM cells at primary diagnosis (n = 8, dots) or relapse (n = 3, rectangle) were incubated for 48 h with PAT-SM6 or isotype in media containing IL-6 and 10% FCS. Dead cells were determined by FACS analysis (Propidium iodide/LDS75 double positive cells). As control, PBMCs from healthy volunteers were treated under the same conditions. In contrast to controls PAT-SM6 significantly induced programmed cell death. C, MM1.S cells were incubated with PAT-SM6 or controls (staurophorin as positive control) for 72 h. AnnexinV and 7AAD positive cells were assessed by flow cytometry. Percentage of 7-AAD/Annexin V double positive cells, Isotype: 14%, PAT-SM6∶79% and positive control: 76%. D, MM1.S cells were incubated with PAT-SM6 or controls (staurophorin or isotype) for 6 h and caspase 3 activation was measured using the <t>AC-DEVD-AMC</t> protease assay. E, Bone marrow cells from a MM patient were CD33-purified to obtain myeloid progenitors and stimulated with PAT-SM6 or controls. No specific binding and subsequently no specific cytotoxicity was observed. F, Cells from the same patient as in E were CD138-isolated and incubated with PAT-SM6 (200 µg/mL) for 24 h. Specific PAT-SM6 binding and killing was observed.
    Fluorophore Substrate Ac Devd Amc, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore fluorogenic tetrapeptide ac devd amc
    In vitro inhibition of caspase-3 activity by AMVIAP. Human caspase-9 activity was assayed in vitro with <t>Ac-DEVD-AMC</t> as the substrate. Caspase-3 was mixed with either, in the absence of recombinant IAPs (▪), GST at 2 μM (•), GST-XIAP at 4.1 μg (500 nM final) (+), or GST-AMVIAP at 2.9 μg (500 nM) (○). Activity was estimated by DEVD-AMC cleavage. Each data series represents fluorescence emission (490 nm) at 1-min intervals for 120 min.
    Fluorogenic Tetrapeptide Ac Devd Amc, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson peptide substrate ac devd amc
    In vitro inhibition of caspase-3 activity by AMVIAP. Human caspase-9 activity was assayed in vitro with <t>Ac-DEVD-AMC</t> as the substrate. Caspase-3 was mixed with either, in the absence of recombinant IAPs (▪), GST at 2 μM (•), GST-XIAP at 4.1 μg (500 nM final) (+), or GST-AMVIAP at 2.9 μg (500 nM) (○). Activity was estimated by DEVD-AMC cleavage. Each data series represents fluorescence emission (490 nm) at 1-min intervals for 120 min.
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    Peptide Institute fluorogenic ac devd amc peptide
    In vitro inhibition of caspase-3 activity by AMVIAP. Human caspase-9 activity was assayed in vitro with <t>Ac-DEVD-AMC</t> as the substrate. Caspase-3 was mixed with either, in the absence of recombinant IAPs (▪), GST at 2 μM (•), GST-XIAP at 4.1 μg (500 nM final) (+), or GST-AMVIAP at 2.9 μg (500 nM) (○). Activity was estimated by DEVD-AMC cleavage. Each data series represents fluorescence emission (490 nm) at 1-min intervals for 120 min.
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    Biomol GmbH peptide substrate ac devd amc
    In vitro inhibition of caspase-3 activity by AMVIAP. Human caspase-9 activity was assayed in vitro with <t>Ac-DEVD-AMC</t> as the substrate. Caspase-3 was mixed with either, in the absence of recombinant IAPs (▪), GST at 2 μM (•), GST-XIAP at 4.1 μg (500 nM final) (+), or GST-AMVIAP at 2.9 μg (500 nM) (○). Activity was estimated by DEVD-AMC cleavage. Each data series represents fluorescence emission (490 nm) at 1-min intervals for 120 min.
    Peptide Substrate Ac Devd Amc, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Enzo Biochem fluorigenic substrate ac devd amc
    Loperamide, pimozide- or STF-62247-induced cell death does not primarily involve apoptosis, ferroptosis, or necroptosis. a , c , d MZ-54 cells were pretreated for 1 h with 20 µM zVAD.fmk ( a ), 5 µM Fer-1 ( c ) or 30 µM Nec-1s ( d ) followed by treatment with 17.5 µM loperamide, 15 µM pimozide, 40 µM STF-62247, 20 µM IM/100 µM TIC, 25 µM ABT-737/100 µM etoposide, 500 nM RSL3 or 1 ng/mL TNF \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\alpha}$$\end{document} α + 0.5 µM BV6 for 48 h. Cell death was assessed by measuring the PI uptake as fraction of total nuclei determined by Hoechst counterstaining using high-content fluorescence microscopy. HT-29 cells served as positive control for induction of necroptotic cell death. b MZ-54 cells were treated with 3 µM STS, 15 µM loperamide, 15 µM pimozide, 40 µM STF-62247, or 20 µM IM/100 µM TIC for the indicated time points. Caspase-3 activity was determined by quantifying alterations in <t>Ac-DEVD-AMC</t> fluorescence. Mean and SEM of 3−4 independent experiments performed in triplicate are shown. * p
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    Millipore proluminescent substrates ac devd amc
    Cell proliferation and caspase-3 activity assay. (A) Stable overexpression of CYP2E1 inhibits the cell growth of HepG2 cells. E47 and C34 cells, two stably transfected HepG2 cell lines harboring CYP2E1 recombinant gene or its empty vector respectively, were seeded with the same number in triplicate on 96-well plates and cultured for up to 6 days. Each day, the triplicate of cultured cells was harvested and subjected to CCK-8 assay at 450 nm. The amount of the formazan dye, generated by the activities of dehydrogenases in cells, is directly proportional to the number of living cells. (B) Activities of caspase-3 increased in HepG2 cells which stably expresses ectopic CYP2E1. E47 and C34 cells were seeded in triplicate on 96-well plates. Cells were harvested on day 1, 3 and 6, and cell lysates were used to test the activities of caspase-3 by measuring proteolytic cleavage of the <t>proluminescent</t> substrates <t>AC-DEVD-AMC.</t> The fluorescence was detected to reflect the amount of released AMC (caspase-3, λex = 380, λem = 460). The results were expressed as arbitrary units of fluorescence (AUF) per milligram of lysate protein.
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    Beyotime ac devd amc
    Cell proliferation and caspase-3 activity assay. (A) Stable overexpression of CYP2E1 inhibits the cell growth of HepG2 cells. E47 and C34 cells, two stably transfected HepG2 cell lines harboring CYP2E1 recombinant gene or its empty vector respectively, were seeded with the same number in triplicate on 96-well plates and cultured for up to 6 days. Each day, the triplicate of cultured cells was harvested and subjected to CCK-8 assay at 450 nm. The amount of the formazan dye, generated by the activities of dehydrogenases in cells, is directly proportional to the number of living cells. (B) Activities of caspase-3 increased in HepG2 cells which stably expresses ectopic CYP2E1. E47 and C34 cells were seeded in triplicate on 96-well plates. Cells were harvested on day 1, 3 and 6, and cell lysates were used to test the activities of caspase-3 by measuring proteolytic cleavage of the <t>proluminescent</t> substrates <t>AC-DEVD-AMC.</t> The fluorescence was detected to reflect the amount of released AMC (caspase-3, λex = 380, λem = 460). The results were expressed as arbitrary units of fluorescence (AUF) per milligram of lysate protein.
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    Hitachi Ltd ac devd amc
    Cell proliferation and caspase-3 activity assay. (A) Stable overexpression of CYP2E1 inhibits the cell growth of HepG2 cells. E47 and C34 cells, two stably transfected HepG2 cell lines harboring CYP2E1 recombinant gene or its empty vector respectively, were seeded with the same number in triplicate on 96-well plates and cultured for up to 6 days. Each day, the triplicate of cultured cells was harvested and subjected to CCK-8 assay at 450 nm. The amount of the formazan dye, generated by the activities of dehydrogenases in cells, is directly proportional to the number of living cells. (B) Activities of caspase-3 increased in HepG2 cells which stably expresses ectopic CYP2E1. E47 and C34 cells were seeded in triplicate on 96-well plates. Cells were harvested on day 1, 3 and 6, and cell lysates were used to test the activities of caspase-3 by measuring proteolytic cleavage of the <t>proluminescent</t> substrates <t>AC-DEVD-AMC.</t> The fluorescence was detected to reflect the amount of released AMC (caspase-3, λex = 380, λem = 460). The results were expressed as arbitrary units of fluorescence (AUF) per milligram of lysate protein.
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    Peptides International ac devd amc
    Op-IAP 1–216 acceleration of caspase activation. SF21 cells were UV irradiated 18 h after transfection with a plasmid encoding Op-IAP HA/1–216 (solid bars) or the vector alone (striped bars). Cell extracts prepared immediately prior to irradiation (0 h) or at the indicated times after irradiation were assayed for caspase activity by using <t>Ac-DEVD-AMC</t> as the substrate. Relative caspase activities are averages ± the standard deviations of triplicate assays. Caspase activity in extracts from stably transfected Op-IAP cell line E6 after UV irradiation (open bars) was included.
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    Axxora ac devd amc
    ( A – J ) Biocompatibility of C–Co–SiO 2 and C–Co 3 O 4 –SiO 2 with lung respiration, adenosine triphosphate (ATP) content, caspase activity, and histology. ( A and B ) Lung specimens were incubated in vitro at 37°C in oxygenated Krebs–Henseleit (KH) buffer with and without 0.2 mg/dL C–Co–SiO 2 or C–Co 3 O 4 –SiO 2 . A summary of the values of k c (μM O 2 /minute/mg) for all experiments is shown in ( A ). The P -values are for comparisons with the untreated condition; “n” is the number of independent measurements. Representative high-performance liquid chromatography (HPLC) runs at 4 hours are shown in ( B ). Briefly, lung specimens were incubated at 37°C with 37 <t>Ac-DEVD-AMC</t> μM for 20 minutes. The tissues were then disrupted by vigorous homogenization, and the supernatants were separated on HPLC and analyzed for the free AMC moiety. Ac-DEVD-AMC had a retention time, R t , of ~4.5 minutes (insert) and AMC of ~12.7 minutes (running solvents, CH 3 CN:dH 2 O 1:3). ( C – F ) Lung specimens were collected 60 minutes after intratracheal instillation of 10 mg C–Co–SiO 2 , 10 mg C–Co 3 O 4 –SiO 2 , or 150 μL 0.9% NaCl. The samples were then incubated in vitro at 37°C in oxygenated KH buffer. Cellular respiration and caspase activity were measured as a function of time. Representative O 2 runs are show in ( C and D ); the values of k c (μM O 2 /minute/mg) are shown at the bottom of each run. Representative HPLC runs of the samples 4 hours after incubation of the specimen with the nanoparticles are shown in ( E ). Ac-DEVD-AMC had a retention time, R t , of ~2.5 minutes (insert) and AMC of ~4.7 minutes (running solvents, methanol:dH 2 O 1:1). The AMC peak area is plotted as a function of time in ( F ). ( G – J ) Representative histology (hematoxylin and eosin) 4 hours after in vitro treatment ( G and H ) and intratracheally treated ( I and J ) lungs with C–Co–SiO 2 ( G and I ), or C–Co 3 O 4 –SiO 2 ( H and J ). The in vitro treatment with C–Co–SiO 2 ( G , 40×) revealed necrosis of the alveolar wall (arrow) and numerous apoptotic bodies (arrowheads), compared with a better-preserved area of the alveolar wall. Similar alterations were noted with C–Co 3 O 4 –SiO 2 ( H , 20×). The in vivo treatment with C–Co–SiO 2 revealed focal areas of alveolar wall damage and apoptotic bodies (arrows) adjacent to bronchioles. ( I , 20×). The in vivo treatment with C–Co 3 O 4 –SiO 2 revealed pulmonary tissue with preserved architecture. The alveolar wall is thinned, and there is vacuolization of pneumocytes and endothelial cells, with loss of definite cell outlines ( J , 20×).
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    Bachem ac devd amc
    D609 enhances FasL-induced caspase-independent ceramide increase and cell death. Jurkat cells (clone A3) were pre-incubated in the presence or absence of 40 μM zVAD-fmk for 1 h and further incubated with or without FasL (500 ng/mL) for 16 h or the indicated times. Ceramide level (expressed as nmol of ceramide per mg of protein) ( A ) and caspase activity toward <t>Ac-DEVD-AMC</t> ( B ) were measured. ( C ) Cells were pre-incubated for 1 h with 40 μM zVAD-fmk (black bars) or a combination of 40 μM zVAD-fmk and 50 μg/mL D609 (white bars). Cells were further incubated with 500 ng/mL FasL for the indicated times and ceramide concentration was measured. Data are expressed as the percentage of values measured in cells incubated with zVAD-fmk alone. ( A – C ) Values are means ± S.E.M. of three independent experiments. ( D ) Cells were pre-incubated for 1 h with 40 μM zVAD-fmk in the presence (triangles) or absence (squares) of 50 μg/mL D609. Cells were further incubated for the indicated times with (solid symbols) or without (empty symbols) 500 ng/mL FasL. Cell death was evaluated by flow cytometry after annexin-V-FITC and propidium iodide labeling. Under these conditions, most of the dead cells were labeled by both annexin-V-FITC and propidium iodide. Data are representative of two independent experiments.
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    AG Scientific ac devd amc
    D609 enhances FasL-induced caspase-independent ceramide increase and cell death. Jurkat cells (clone A3) were pre-incubated in the presence or absence of 40 μM zVAD-fmk for 1 h and further incubated with or without FasL (500 ng/mL) for 16 h or the indicated times. Ceramide level (expressed as nmol of ceramide per mg of protein) ( A ) and caspase activity toward <t>Ac-DEVD-AMC</t> ( B ) were measured. ( C ) Cells were pre-incubated for 1 h with 40 μM zVAD-fmk (black bars) or a combination of 40 μM zVAD-fmk and 50 μg/mL D609 (white bars). Cells were further incubated with 500 ng/mL FasL for the indicated times and ceramide concentration was measured. Data are expressed as the percentage of values measured in cells incubated with zVAD-fmk alone. ( A – C ) Values are means ± S.E.M. of three independent experiments. ( D ) Cells were pre-incubated for 1 h with 40 μM zVAD-fmk in the presence (triangles) or absence (squares) of 50 μg/mL D609. Cells were further incubated for the indicated times with (solid symbols) or without (empty symbols) 500 ng/mL FasL. Cell death was evaluated by flow cytometry after annexin-V-FITC and propidium iodide labeling. Under these conditions, most of the dead cells were labeled by both annexin-V-FITC and propidium iodide. Data are representative of two independent experiments.
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    Enzo Biochem fluorogenic substrate ac devd amc
    D609 enhances FasL-induced caspase-independent ceramide increase and cell death. Jurkat cells (clone A3) were pre-incubated in the presence or absence of 40 μM zVAD-fmk for 1 h and further incubated with or without FasL (500 ng/mL) for 16 h or the indicated times. Ceramide level (expressed as nmol of ceramide per mg of protein) ( A ) and caspase activity toward <t>Ac-DEVD-AMC</t> ( B ) were measured. ( C ) Cells were pre-incubated for 1 h with 40 μM zVAD-fmk (black bars) or a combination of 40 μM zVAD-fmk and 50 μg/mL D609 (white bars). Cells were further incubated with 500 ng/mL FasL for the indicated times and ceramide concentration was measured. Data are expressed as the percentage of values measured in cells incubated with zVAD-fmk alone. ( A – C ) Values are means ± S.E.M. of three independent experiments. ( D ) Cells were pre-incubated for 1 h with 40 μM zVAD-fmk in the presence (triangles) or absence (squares) of 50 μg/mL D609. Cells were further incubated for the indicated times with (solid symbols) or without (empty symbols) 500 ng/mL FasL. Cell death was evaluated by flow cytometry after annexin-V-FITC and propidium iodide labeling. Under these conditions, most of the dead cells were labeled by both annexin-V-FITC and propidium iodide. Data are representative of two independent experiments.
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    AnaSpec fluorogenic substrate ac devd amc
    D609 enhances FasL-induced caspase-independent ceramide increase and cell death. Jurkat cells (clone A3) were pre-incubated in the presence or absence of 40 μM zVAD-fmk for 1 h and further incubated with or without FasL (500 ng/mL) for 16 h or the indicated times. Ceramide level (expressed as nmol of ceramide per mg of protein) ( A ) and caspase activity toward <t>Ac-DEVD-AMC</t> ( B ) were measured. ( C ) Cells were pre-incubated for 1 h with 40 μM zVAD-fmk (black bars) or a combination of 40 μM zVAD-fmk and 50 μg/mL D609 (white bars). Cells were further incubated with 500 ng/mL FasL for the indicated times and ceramide concentration was measured. Data are expressed as the percentage of values measured in cells incubated with zVAD-fmk alone. ( A – C ) Values are means ± S.E.M. of three independent experiments. ( D ) Cells were pre-incubated for 1 h with 40 μM zVAD-fmk in the presence (triangles) or absence (squares) of 50 μg/mL D609. Cells were further incubated for the indicated times with (solid symbols) or without (empty symbols) 500 ng/mL FasL. Cell death was evaluated by flow cytometry after annexin-V-FITC and propidium iodide labeling. Under these conditions, most of the dead cells were labeled by both annexin-V-FITC and propidium iodide. Data are representative of two independent experiments.
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    Image Search Results


    BAX is required for increases in caspase activity. Cultures were switched to K5+S medium, lysed after 4, 8, 12, 24, 48, or 72 h, and cleavage of DEVD-AMC was determined. Control cultures were switched to K25+S medium and treated identically. Data represent mean ± SD for triplicate measurements from one experiment and are representative of two additional independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: Bax Deletion Further Orders the Cell Death Pathway in Cerebellar Granule Cells and Suggests a Caspase-independent Pathway to Cell Death

    doi:

    Figure Lengend Snippet: BAX is required for increases in caspase activity. Cultures were switched to K5+S medium, lysed after 4, 8, 12, 24, 48, or 72 h, and cleavage of DEVD-AMC was determined. Control cultures were switched to K25+S medium and treated identically. Data represent mean ± SD for triplicate measurements from one experiment and are representative of two additional independent experiments.

    Article Snippet: In a 96-well plate, 25 μl of lysate was combined with 75 μl of buffer B (25 mM Hepes, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, 3 mM DTT, pH 7.5) containing 30 μM Ac-DEVD-AMC (Biomol Research Laboratories, Plymouth, PA) and incubated for 20 min at room temperature in the dark.

    Techniques: Activity Assay

    Inhibitors of caspases do not block death, but do block DEVD-AMC cleavage. Cultures were switched to K5+S, K5+S plus 100 μM BAF, or K5+S plus 100 μM ZVAD-FMK. Control cultures were switched to K25+S medium. ( A ) After 12, 24, or 48 h neuronal survival was determined by calcein AM staining. (mean ± SD, N = 3 experiments) ( B ) After 8, 12, 18, 24, or 48 h cultures were lysed and assayed for DEVD-AMC cleavage. Fluorescence was measured after 20 min at room temperature (mean ± range, N = 2 experiments).

    Journal: The Journal of Cell Biology

    Article Title: Bax Deletion Further Orders the Cell Death Pathway in Cerebellar Granule Cells and Suggests a Caspase-independent Pathway to Cell Death

    doi:

    Figure Lengend Snippet: Inhibitors of caspases do not block death, but do block DEVD-AMC cleavage. Cultures were switched to K5+S, K5+S plus 100 μM BAF, or K5+S plus 100 μM ZVAD-FMK. Control cultures were switched to K25+S medium. ( A ) After 12, 24, or 48 h neuronal survival was determined by calcein AM staining. (mean ± SD, N = 3 experiments) ( B ) After 8, 12, 18, 24, or 48 h cultures were lysed and assayed for DEVD-AMC cleavage. Fluorescence was measured after 20 min at room temperature (mean ± range, N = 2 experiments).

    Article Snippet: In a 96-well plate, 25 μl of lysate was combined with 75 μl of buffer B (25 mM Hepes, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, 3 mM DTT, pH 7.5) containing 30 μM Ac-DEVD-AMC (Biomol Research Laboratories, Plymouth, PA) and incubated for 20 min at room temperature in the dark.

    Techniques: Blocking Assay, Staining, Fluorescence

    Cleavage of GCLC and depletion of cellular GSH during apoptotic cell death. HeLa cells were treated with human TNF (30 ng/ml) and cycloheximide (CHX, 10 μg/ml) for the time period indicated. A: GCLC, GCLM, and pro-caspase-3 were analyzed by immunoblotting. Apoptosis was quantified by assessing apoptotic nuclear morphology of DAPI-stained cells. B: Cellular glutathione levels (GSH+GSSG; closed bars Caspase-3-like activities ( open bars ) were determined using the fluorogenic DEVD-AMC substrate. Data presented are from a representative experiment performed at least three times with similar results.

    Journal: The American Journal of Pathology

    Article Title: Caspase-3-Dependent Cleavage of the Glutamate-l-Cysteine Ligase Catalytic Subunit during Apoptotic Cell Death

    doi:

    Figure Lengend Snippet: Cleavage of GCLC and depletion of cellular GSH during apoptotic cell death. HeLa cells were treated with human TNF (30 ng/ml) and cycloheximide (CHX, 10 μg/ml) for the time period indicated. A: GCLC, GCLM, and pro-caspase-3 were analyzed by immunoblotting. Apoptosis was quantified by assessing apoptotic nuclear morphology of DAPI-stained cells. B: Cellular glutathione levels (GSH+GSSG; closed bars Caspase-3-like activities ( open bars ) were determined using the fluorogenic DEVD-AMC substrate. Data presented are from a representative experiment performed at least three times with similar results.

    Article Snippet: Ac-DEVD-AMC was from Alexis Biochemicals (San Diego, CA), Ac-DEVD-CHO was from Biomol (Plymouth Meeting, PA), and z-VAD-fmk was from Bachem (King of Prussia, PA).

    Techniques: Staining

    Inhibition of the ERK1/2 pathway blocks BDNF-mediated inhibition of caspase-3-like activity. P7 rats received ICV injections of vehicle or U0126 (1 nmol/animal) followed 15 min later by an ICV injection of vehicle or BDNF (5 μg/animal) just before the carotid ligation and exposure to 2.5 hr of hypoxia. Twenty-four hours after H-I, brain tissues from the hippocampus ( A ) and cortex ( B ) contralateral ( right ) and ipsilateral ( left ) to the ligation were dissected and subjected to DEVD-AMC cleavage assay as described in Materials and Methods. Note that there is no activation of caspase-3-like activity in the group treated with U0126 without subsequent H-I ( No HI ). Data represent the mean ± SEM; * p

    Journal: The Journal of Neuroscience

    Article Title: BDNF Protects the Neonatal Brain from Hypoxic-Ischemic InjuryIn Vivo via the ERK Pathway

    doi: 10.1523/JNEUROSCI.20-15-05775.2000

    Figure Lengend Snippet: Inhibition of the ERK1/2 pathway blocks BDNF-mediated inhibition of caspase-3-like activity. P7 rats received ICV injections of vehicle or U0126 (1 nmol/animal) followed 15 min later by an ICV injection of vehicle or BDNF (5 μg/animal) just before the carotid ligation and exposure to 2.5 hr of hypoxia. Twenty-four hours after H-I, brain tissues from the hippocampus ( A ) and cortex ( B ) contralateral ( right ) and ipsilateral ( left ) to the ligation were dissected and subjected to DEVD-AMC cleavage assay as described in Materials and Methods. Note that there is no activation of caspase-3-like activity in the group treated with U0126 without subsequent H-I ( No HI ). Data represent the mean ± SEM; * p

    Article Snippet: Tissue lysates (10 μl) were incubated in a 96-well plate with 90 μl of assay buffer (10 m m HEPES, pH 7.4, 42 m m KCl, 5 m m MgCl2 , 1 m m DTT, and 10% sucrose) containing 30 μ m acetyl-DEVD-AMC (Calbiochem).

    Techniques: Inhibition, Activity Assay, Injection, Ligation, Cleavage Assay, Activation Assay

    Inhibition of the AKT pathway does not block BDNF-mediated neuroprotection against neonatal H-I. A, B , P7 rats received ICV injections of VEH or wortmannin (0.1 nmol/animal) followed 15 min later by ICV injections of VEH or BDNF (5 μg/animal) before the carotid ligation and exposure to hypoxia for 2.5 hr. Twenty-four hours after H-I, brain tissues from the hippocampus ( A ) and cortex ( B ) contralateral ( right ) and ipsilateral ( left ) to the ligation were dissected and subjected to DEVD-AMC cleavage assay as described in Materials and Methods. Note that there is no activation of caspase-3-like activity in the group treated with wortmannin without subsequent H-I. Data represent the mean ± SEM; * p

    Journal: The Journal of Neuroscience

    Article Title: BDNF Protects the Neonatal Brain from Hypoxic-Ischemic InjuryIn Vivo via the ERK Pathway

    doi: 10.1523/JNEUROSCI.20-15-05775.2000

    Figure Lengend Snippet: Inhibition of the AKT pathway does not block BDNF-mediated neuroprotection against neonatal H-I. A, B , P7 rats received ICV injections of VEH or wortmannin (0.1 nmol/animal) followed 15 min later by ICV injections of VEH or BDNF (5 μg/animal) before the carotid ligation and exposure to hypoxia for 2.5 hr. Twenty-four hours after H-I, brain tissues from the hippocampus ( A ) and cortex ( B ) contralateral ( right ) and ipsilateral ( left ) to the ligation were dissected and subjected to DEVD-AMC cleavage assay as described in Materials and Methods. Note that there is no activation of caspase-3-like activity in the group treated with wortmannin without subsequent H-I. Data represent the mean ± SEM; * p

    Article Snippet: Tissue lysates (10 μl) were incubated in a 96-well plate with 90 μl of assay buffer (10 m m HEPES, pH 7.4, 42 m m KCl, 5 m m MgCl2 , 1 m m DTT, and 10% sucrose) containing 30 μ m acetyl-DEVD-AMC (Calbiochem).

    Techniques: Inhibition, Blocking Assay, Ligation, Cleavage Assay, Activation Assay, Activity Assay

    Renal caspase-like activities were determined kinetically in homogenates of tissue obtained after 24 hours ( a and c ) and 2 hours ( b and d ) of renal reperfusion in a fluorogenic substrate assay in which Ac-YVAD-amc (caspase-1–like) ( a and b ) or Ac-DEVD-amc (caspase-3–like) ( c and d ) served as substrates. Note that these data may represent a more generalized form of caspase activation, as outlined in the main text. Data are expressed as the increase in fluorescence as a function of time, normalized against data obtained from the sham-operated group. The groups that indicate t = 2 on the x-axis received the indicated treatment after 2 hours of reperfusion. All other groups were treated at the time of reperfusion. * P

    Journal: Journal of Clinical Investigation

    Article Title: Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation

    doi:

    Figure Lengend Snippet: Renal caspase-like activities were determined kinetically in homogenates of tissue obtained after 24 hours ( a and c ) and 2 hours ( b and d ) of renal reperfusion in a fluorogenic substrate assay in which Ac-YVAD-amc (caspase-1–like) ( a and b ) or Ac-DEVD-amc (caspase-3–like) ( c and d ) served as substrates. Note that these data may represent a more generalized form of caspase activation, as outlined in the main text. Data are expressed as the increase in fluorescence as a function of time, normalized against data obtained from the sham-operated group. The groups that indicate t = 2 on the x-axis received the indicated treatment after 2 hours of reperfusion. All other groups were treated at the time of reperfusion. * P

    Article Snippet: Guler (Chiron Corp., Emeryville, California, USA); ZVAD-fmk and ZFA-fmk were obtained from Enzyme Systems Products Inc. (Livermore, California, USA); Ac-YVAD-amc and Ac-DEVD-amc were obtained from the Peptide Institute (Osaka, Japan).

    Techniques: Activation Assay, Fluorescence

    Comparative analysis of 5-FU-induced apoptosis in wt and p53-deficient HCT116 cells The apoptotic indicators, cleaved PARP and lamin A (cle PARP, cle lamin A), were analyzed at multiple time points ranging from 0 to 36 h post-treatment initiation in p53 -proficient and 0 to 48 h in p53 -deficient HCT116 cells using immunoblotting of SDS-PAGE-separated cell lysates A. Caspase-3/-7-like activities were measured by monitoring the liberation of AMC from the synthetic fluoro-conjugated peptide substrate motif DEVD (DEVD-AMC) B. Drug effects with respect to loss of mitochondrial membrane potential (ΔΨ m ) in live cells were examined by FACS analysis of mitochondrial TMRE accumulation C. HCT116 wt and p53 −/− cells, treated with 768 μM 5-FU for time periods indicated, were harvested and subjected to separation into cytoplasmic and nuclear/mitochondrial fractions, enabling immunoblotting of released cytochrome c D. Cells, treated with 5-FU (10 μM, 48 h or 768 μM, 24 h), were fixed in 70% ethanol and stained with propidium iodide before FACS determination of sub G1 populations E. Clonal survival of eighteen HCT116 wt or p53 −/− cells/cm 2 in response to 5-FU treatment (48 h), ranging from 10 to 50 μM F. GAPDH served as a marker for equal sample loading in (A) and (D), and also as an indicator of fractionation efficacy in (D).

    Journal: Oncotarget

    Article Title: 5-Fluorouracil-induced RNA stress engages a TRAIL-DISC-dependent apoptosis axis facilitated by p53

    doi:

    Figure Lengend Snippet: Comparative analysis of 5-FU-induced apoptosis in wt and p53-deficient HCT116 cells The apoptotic indicators, cleaved PARP and lamin A (cle PARP, cle lamin A), were analyzed at multiple time points ranging from 0 to 36 h post-treatment initiation in p53 -proficient and 0 to 48 h in p53 -deficient HCT116 cells using immunoblotting of SDS-PAGE-separated cell lysates A. Caspase-3/-7-like activities were measured by monitoring the liberation of AMC from the synthetic fluoro-conjugated peptide substrate motif DEVD (DEVD-AMC) B. Drug effects with respect to loss of mitochondrial membrane potential (ΔΨ m ) in live cells were examined by FACS analysis of mitochondrial TMRE accumulation C. HCT116 wt and p53 −/− cells, treated with 768 μM 5-FU for time periods indicated, were harvested and subjected to separation into cytoplasmic and nuclear/mitochondrial fractions, enabling immunoblotting of released cytochrome c D. Cells, treated with 5-FU (10 μM, 48 h or 768 μM, 24 h), were fixed in 70% ethanol and stained with propidium iodide before FACS determination of sub G1 populations E. Clonal survival of eighteen HCT116 wt or p53 −/− cells/cm 2 in response to 5-FU treatment (48 h), ranging from 10 to 50 μM F. GAPDH served as a marker for equal sample loading in (A) and (D), and also as an indicator of fractionation efficacy in (D).

    Article Snippet: Measurement of caspase-3/-7-like activities Measurement of the caspase-3/-7-like substrate Ac-DEVD-AMC (acetyl Asp-Glu-Val-Asp 7-amido- 4-methylcoumarin; Peptide Institute, Osaka, Japan) cleavage was performed as described [ ].

    Techniques: SDS Page, FACS, Staining, Marker, Fractionation

    N. gonorrhoeae inhibits extrinsic apoptosis induced by TRAIL. (A) N. gonorrhoeae inhibits DNA fragmentation in HL-60 induced by TRAIL. Differentiated HL-60 cells were infected with piliated, Opa-expressing FA1090 at an MOI of 9 and then treated with 100 ng/ml TRAIL. DNA fragmentation was evaluated by flow cytometry, and the percentage of cells exhibiting hypodiploid DNA under each condition is indicated. (B) Average percentage of cells exhibiting DNA fragmentation after infection and/or treatment with 100 ng/ml TRAIL. The data are averages for 3 independent experiments. (C) N. gonorrhoeae infection inhibits TRAIL-induced caspase-3 activity in HL-60 cell lysates. HL-60 cells were infected with piliated strain FA1090 at an average MOI of 15 and then treated with 100 ng/ml TRAIL. Caspase-3 activity was measured using Ac-DEVD-AMC. Data are presented as the relative caspase-3 activity compared to that of uninfected, untreated controls and are representative of 6 independent biological replicates.

    Journal: Infection and Immunity

    Article Title: Neisseria gonorrhoeae-Mediated Inhibition of Apoptotic Signalling in Polymorphonuclear Leukocytes ▿

    doi: 10.1128/IAI.01267-10

    Figure Lengend Snippet: N. gonorrhoeae inhibits extrinsic apoptosis induced by TRAIL. (A) N. gonorrhoeae inhibits DNA fragmentation in HL-60 induced by TRAIL. Differentiated HL-60 cells were infected with piliated, Opa-expressing FA1090 at an MOI of 9 and then treated with 100 ng/ml TRAIL. DNA fragmentation was evaluated by flow cytometry, and the percentage of cells exhibiting hypodiploid DNA under each condition is indicated. (B) Average percentage of cells exhibiting DNA fragmentation after infection and/or treatment with 100 ng/ml TRAIL. The data are averages for 3 independent experiments. (C) N. gonorrhoeae infection inhibits TRAIL-induced caspase-3 activity in HL-60 cell lysates. HL-60 cells were infected with piliated strain FA1090 at an average MOI of 15 and then treated with 100 ng/ml TRAIL. Caspase-3 activity was measured using Ac-DEVD-AMC. Data are presented as the relative caspase-3 activity compared to that of uninfected, untreated controls and are representative of 6 independent biological replicates.

    Article Snippet: To measure caspase-3 activity in the cell lysates, 5 μl reconstituted caspase-3 substrate at a concentration of 1.0 mg/ml (Ac-DEVD-AMC; BD Pharmingen) was incubated with 0.2 ml HEPES buffer (20 mM HEPES [pH 7.5], 10% glycerol, 2 mM dithiothreitol [DTT]) and 25 μl cell lysates for 60 min at 37°C.

    Techniques: Infection, Expressing, Flow Cytometry, Cytometry, Activity Assay

    N. gonorrhoeae inhibits caspase-3 activity induced by STS in HL-60 cells. (A) N. gonorrhoeae inhibits caspase-3 activity in HL-60 cell lysates. Differentiated HL-60 cells were infected with piliated strain FA1090 at an average MOI of 61 and then treated with 1 μM STS, 20 μM z-VAD-fmk, or DMSO. Caspase-3 activity was measured using the fluorogenic substrate Ac-DEVD-AMC, and data are presented as the caspase-3 activation relative to that for uninfected, DMSO-treated controls. Data are representative of at least 3 biological replicates. (B) N. gonorrhoeae inhibits procaspase-3 cleavage in STS-treated cells. Differentiated HL-60 cells were infected at an MOI of 27 and then treated with 1 μM STS. Cell lysates were harvested and subjected to Western blotting using an antibody directed against full-length procaspase-3. Data are representative of 3 independent experiments. (C) Inhibition of STS-induced caspase-3 activity by N. gonorrhoeae is dose dependent. HL-60 cells were infected with N. gonorrhoeae at increasing MOIs and then treated with STS. Data are representative of at least 3 independent experiments. (D) Inhibition of STS-induced caspase-3 activity requires live bacteria. HL-60 cells were infected with live (average MOI = 60) or heat-killed bacteria, followed by treatment with STS, after which caspase-3 activity in cell lysates was assessed. The data are representative of 4 independent experiments.

    Journal: Infection and Immunity

    Article Title: Neisseria gonorrhoeae-Mediated Inhibition of Apoptotic Signalling in Polymorphonuclear Leukocytes ▿

    doi: 10.1128/IAI.01267-10

    Figure Lengend Snippet: N. gonorrhoeae inhibits caspase-3 activity induced by STS in HL-60 cells. (A) N. gonorrhoeae inhibits caspase-3 activity in HL-60 cell lysates. Differentiated HL-60 cells were infected with piliated strain FA1090 at an average MOI of 61 and then treated with 1 μM STS, 20 μM z-VAD-fmk, or DMSO. Caspase-3 activity was measured using the fluorogenic substrate Ac-DEVD-AMC, and data are presented as the caspase-3 activation relative to that for uninfected, DMSO-treated controls. Data are representative of at least 3 biological replicates. (B) N. gonorrhoeae inhibits procaspase-3 cleavage in STS-treated cells. Differentiated HL-60 cells were infected at an MOI of 27 and then treated with 1 μM STS. Cell lysates were harvested and subjected to Western blotting using an antibody directed against full-length procaspase-3. Data are representative of 3 independent experiments. (C) Inhibition of STS-induced caspase-3 activity by N. gonorrhoeae is dose dependent. HL-60 cells were infected with N. gonorrhoeae at increasing MOIs and then treated with STS. Data are representative of at least 3 independent experiments. (D) Inhibition of STS-induced caspase-3 activity requires live bacteria. HL-60 cells were infected with live (average MOI = 60) or heat-killed bacteria, followed by treatment with STS, after which caspase-3 activity in cell lysates was assessed. The data are representative of 4 independent experiments.

    Article Snippet: To measure caspase-3 activity in the cell lysates, 5 μl reconstituted caspase-3 substrate at a concentration of 1.0 mg/ml (Ac-DEVD-AMC; BD Pharmingen) was incubated with 0.2 ml HEPES buffer (20 mM HEPES [pH 7.5], 10% glycerol, 2 mM dithiothreitol [DTT]) and 25 μl cell lysates for 60 min at 37°C.

    Techniques: Activity Assay, Infection, Activation Assay, Western Blot, Inhibition

    N. gonorrhoeae infection inhibits spontaneous apoptosis in primary PMNs but does not fully inhibit STS-induced apoptosis. (A) N. gonorrhoeae infection inhibits DNA fragmentation in PMNs undergoing spontaneous apoptosis. Primary PMNs isolated from healthy human volunteers were infected at an MOI of 44 and were treated with 1 μM STS overnight. DNA fragmentation was evaluated by flow cytometry. (B) Average percentage of cells exhibiting DNA fragmentation after infection at an MOI of ∼50 and/or treatment with STS/DMSO. The data are representative of 6 independent experiments. (C) N. gonorrhoeae infection inhibits caspase-3 activity in PMNs undergoing both spontaneous and STS-induced apoptosis. Primary PMNs isolated from healthy human volunteers were infected at increasing MOIs and then treated with STS. Caspase-3 activity in cell lysates was measured using Ac-DEVD-AMC. Data are presented as the relative caspase-3 activity compared to that of uninfected, untreated controls and are representative of at least 8 independent biological replicates.

    Journal: Infection and Immunity

    Article Title: Neisseria gonorrhoeae-Mediated Inhibition of Apoptotic Signalling in Polymorphonuclear Leukocytes ▿

    doi: 10.1128/IAI.01267-10

    Figure Lengend Snippet: N. gonorrhoeae infection inhibits spontaneous apoptosis in primary PMNs but does not fully inhibit STS-induced apoptosis. (A) N. gonorrhoeae infection inhibits DNA fragmentation in PMNs undergoing spontaneous apoptosis. Primary PMNs isolated from healthy human volunteers were infected at an MOI of 44 and were treated with 1 μM STS overnight. DNA fragmentation was evaluated by flow cytometry. (B) Average percentage of cells exhibiting DNA fragmentation after infection at an MOI of ∼50 and/or treatment with STS/DMSO. The data are representative of 6 independent experiments. (C) N. gonorrhoeae infection inhibits caspase-3 activity in PMNs undergoing both spontaneous and STS-induced apoptosis. Primary PMNs isolated from healthy human volunteers were infected at increasing MOIs and then treated with STS. Caspase-3 activity in cell lysates was measured using Ac-DEVD-AMC. Data are presented as the relative caspase-3 activity compared to that of uninfected, untreated controls and are representative of at least 8 independent biological replicates.

    Article Snippet: To measure caspase-3 activity in the cell lysates, 5 μl reconstituted caspase-3 substrate at a concentration of 1.0 mg/ml (Ac-DEVD-AMC; BD Pharmingen) was incubated with 0.2 ml HEPES buffer (20 mM HEPES [pH 7.5], 10% glycerol, 2 mM dithiothreitol [DTT]) and 25 μl cell lysates for 60 min at 37°C.

    Techniques: Infection, Isolation, Flow Cytometry, Cytometry, Activity Assay

    Nuclear factor (NF)-κB signalling in ultraviolet (UV)-irradiated melanocytes. Melanocytes were exposed to UVA (60 J cm −2 ) or UVB (500 mJ cm −2 ). (a) Immunostaining to determine the localization of the p50 and p65 subunits of the NF-κB dimer after irradiation. Images are selected to display the characteristic appearance of cytosolic and nuclear location, respectively. (b) One representative Western blot out of four and the corresponding optical density, showing the levels of the p65 subunit in digitonin-extracted cytosolic fractions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Nuclear localization of the (c) p65 and (d) p50 subunits in melanocytes pretreated with an inhibitor of the p50 subunit of the NF-κB dimer (SN50, 20 μmol L −1 ; black bars) 3 h prior to irradiation. (e) Caspase-3 activation was analysed based on the cleavage of the substrate Ac-DEVD-AMC, 16 h postirradiation [expressed as arbitary units (AU) per mg protein and hour], and (f) apoptosis was quantified via microscopic analysis of nuclear morphology in 4',6-diamidino-2-phenylindole (DAPI)-stained cells after 24 h. UV-induced (g) mitochondrial outer membrane permeabilization (MOMP), as detected by cytochrome c release, and (h) lysosomal membrane permeabilization (LMP), as detected by the release of cathepsin D (cat D). The results are presented as the mean ± SD ( n = 4). * P ≤ 0·05 vs. control.

    Journal: The British Journal of Dermatology

    Article Title: Cell fate regulated by nuclear factor-κB- and activator protein-1-dependent signalling in human melanocytes exposed to ultraviolet A and ultraviolet B

    doi: 10.1111/bjd.13278

    Figure Lengend Snippet: Nuclear factor (NF)-κB signalling in ultraviolet (UV)-irradiated melanocytes. Melanocytes were exposed to UVA (60 J cm −2 ) or UVB (500 mJ cm −2 ). (a) Immunostaining to determine the localization of the p50 and p65 subunits of the NF-κB dimer after irradiation. Images are selected to display the characteristic appearance of cytosolic and nuclear location, respectively. (b) One representative Western blot out of four and the corresponding optical density, showing the levels of the p65 subunit in digitonin-extracted cytosolic fractions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Nuclear localization of the (c) p65 and (d) p50 subunits in melanocytes pretreated with an inhibitor of the p50 subunit of the NF-κB dimer (SN50, 20 μmol L −1 ; black bars) 3 h prior to irradiation. (e) Caspase-3 activation was analysed based on the cleavage of the substrate Ac-DEVD-AMC, 16 h postirradiation [expressed as arbitary units (AU) per mg protein and hour], and (f) apoptosis was quantified via microscopic analysis of nuclear morphology in 4',6-diamidino-2-phenylindole (DAPI)-stained cells after 24 h. UV-induced (g) mitochondrial outer membrane permeabilization (MOMP), as detected by cytochrome c release, and (h) lysosomal membrane permeabilization (LMP), as detected by the release of cathepsin D (cat D). The results are presented as the mean ± SD ( n = 4). * P ≤ 0·05 vs. control.

    Article Snippet: To analyse caspase-3 activity, the cells were collected in lysis buffer (10 mmol L−1 Tris-HCl at pH 7·5, 130 mmol L−1 NaCl, 1% Triton X-100, 10 mmol L−1 sodium pyrophosphate, 10 mmol L−1 NaH2 PO4 /NaHPO4 ) and incubated with the substrate Ac-DEVD-AMC according to the manufacturer's recommendations (BD Biosciences, San Jose, CA, U.S.A.).

    Techniques: Irradiation, Immunostaining, Western Blot, Activation Assay, Staining

    Activator protein-1 signalling in melanocytes following ultraviolet (UV) exposure. Melanocytes pretreated with JUN small interfering (si)RNA (black bars) 24 h prior to UVA (60 J cm −2 ) or UVB (500 mJ cm −2 ) treatment. Quantification of melanocytes showing nuclear staining of (a) c-Jun and (b) c-Fos 4 h after UV exposure. The images were selected to display the characteristic appearance of the nuclear and cytosolic location, respectively. The protein levels and corresponding optical density of (c) c-Jun and (d) c-Fos in digitonin-extracted cytosolic fractions are shown from one representative Western blot out of four. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (e) Caspase-3 activation was analysed based on the cleavage of the substrate Ac-DEVD-AMC, 16 h postirradiation [expressed as arbitrary unites (AU) per mg protein and hour] and (f) apoptosis was quantified through microscopic inspection of nuclear morphology in 4',6-diamidino-2-phenylindole (DAPI)-stained cells after 24 h. UV-induced (g) mitochondrial outer membrane permeabilization (MOMP), as detected by cytochrome c release, and (h) lysosomal membrane permeabilization (LMP), as detected by the release of cathepsin D (cat D) to the cytosol. The results are presented as the mean ± SD ( n = 4). * P ≤ 0·05 vs. control.

    Journal: The British Journal of Dermatology

    Article Title: Cell fate regulated by nuclear factor-κB- and activator protein-1-dependent signalling in human melanocytes exposed to ultraviolet A and ultraviolet B

    doi: 10.1111/bjd.13278

    Figure Lengend Snippet: Activator protein-1 signalling in melanocytes following ultraviolet (UV) exposure. Melanocytes pretreated with JUN small interfering (si)RNA (black bars) 24 h prior to UVA (60 J cm −2 ) or UVB (500 mJ cm −2 ) treatment. Quantification of melanocytes showing nuclear staining of (a) c-Jun and (b) c-Fos 4 h after UV exposure. The images were selected to display the characteristic appearance of the nuclear and cytosolic location, respectively. The protein levels and corresponding optical density of (c) c-Jun and (d) c-Fos in digitonin-extracted cytosolic fractions are shown from one representative Western blot out of four. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (e) Caspase-3 activation was analysed based on the cleavage of the substrate Ac-DEVD-AMC, 16 h postirradiation [expressed as arbitrary unites (AU) per mg protein and hour] and (f) apoptosis was quantified through microscopic inspection of nuclear morphology in 4',6-diamidino-2-phenylindole (DAPI)-stained cells after 24 h. UV-induced (g) mitochondrial outer membrane permeabilization (MOMP), as detected by cytochrome c release, and (h) lysosomal membrane permeabilization (LMP), as detected by the release of cathepsin D (cat D) to the cytosol. The results are presented as the mean ± SD ( n = 4). * P ≤ 0·05 vs. control.

    Article Snippet: To analyse caspase-3 activity, the cells were collected in lysis buffer (10 mmol L−1 Tris-HCl at pH 7·5, 130 mmol L−1 NaCl, 1% Triton X-100, 10 mmol L−1 sodium pyrophosphate, 10 mmol L−1 NaH2 PO4 /NaHPO4 ) and incubated with the substrate Ac-DEVD-AMC according to the manufacturer's recommendations (BD Biosciences, San Jose, CA, U.S.A.).

    Techniques: Staining, Western Blot, Activation Assay

    Nur77 expression inhibits caspase activation induced by TNF stimulation. ( A ) Caspase-3 activity was examined in vector or Nur77 stable transformants in RelA -/- or TRAF2 -/- cells after TNF stimulation. Caspase-3 activity was measured by the appearance of cleaved caspase-3 in Western blot analyses. ( B ) Fluorometric caspase-3 enzymatic activity was measured by using a specific substrate Ac-DEVD-AMC as described in Methods . ( C ) Caspase-8 enzymatic activity was measured by using the substrate Ac-IETD-AFC as described in Methods .

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Nur77 as a survival factor in tumor necrosis factor signaling

    doi: 10.1073/pnas.0932598100

    Figure Lengend Snippet: Nur77 expression inhibits caspase activation induced by TNF stimulation. ( A ) Caspase-3 activity was examined in vector or Nur77 stable transformants in RelA -/- or TRAF2 -/- cells after TNF stimulation. Caspase-3 activity was measured by the appearance of cleaved caspase-3 in Western blot analyses. ( B ) Fluorometric caspase-3 enzymatic activity was measured by using a specific substrate Ac-DEVD-AMC as described in Methods . ( C ) Caspase-8 enzymatic activity was measured by using the substrate Ac-IETD-AFC as described in Methods .

    Article Snippet: Caspase-3 activity was analyzed by using the substrate Ac-DEVD-AMC (PharMingen) as described by Hughes et al. ( ).

    Techniques: Expressing, Activation Assay, Activity Assay, Plasmid Preparation, Western Blot

    Kinetics of cisplatin-induced DEVDase and caspase 9/ LEHDase activities. 224 cells were treated with 20 μM cisplatin for the indicated time periods in the absence or presence of adeno-dnMEKK. Expression of dnMEKK was induced with DOX at 20 h before cisplatin treatment. Empty bars, cisplatin only; filled bars, cisplatin treatment in the presence of dnMEKK. (A) DEVDase activity on the synthetic substrate Ac-DEVD-AMC was assessed twice for each duplicate sample. The inhibitor DEVD-CHO reduced all activity to background in all parallel samples. Results are shown as fold increase in activity in untreated (control [C]) cells. (B) Caspase 9/LEHDase activity on the synthetic substrate Ac-LEHD-AMC. The inhibitor LEHD-CHO reduced all activity to background in all parallel samples. (C) Cell extracts (30 μg/lane) were analyzed by Western blotting of caspase 9. The 35-kDa cleavage fragment represents the active form. β-TUB., tubulin; CISPL, cisplatin.

    Journal: Molecular and Cellular Biology

    Article Title: Cisplatin Induces the Proapoptotic Conformation of Bak in a ?MEKK1-Dependent Manner

    doi: 10.1128/MCB.21.11.3684-3691.2001

    Figure Lengend Snippet: Kinetics of cisplatin-induced DEVDase and caspase 9/ LEHDase activities. 224 cells were treated with 20 μM cisplatin for the indicated time periods in the absence or presence of adeno-dnMEKK. Expression of dnMEKK was induced with DOX at 20 h before cisplatin treatment. Empty bars, cisplatin only; filled bars, cisplatin treatment in the presence of dnMEKK. (A) DEVDase activity on the synthetic substrate Ac-DEVD-AMC was assessed twice for each duplicate sample. The inhibitor DEVD-CHO reduced all activity to background in all parallel samples. Results are shown as fold increase in activity in untreated (control [C]) cells. (B) Caspase 9/LEHDase activity on the synthetic substrate Ac-LEHD-AMC. The inhibitor LEHD-CHO reduced all activity to background in all parallel samples. (C) Cell extracts (30 μg/lane) were analyzed by Western blotting of caspase 9. The 35-kDa cleavage fragment represents the active form. β-TUB., tubulin; CISPL, cisplatin.

    Article Snippet: In addition to cytochrome c release and DNA-dependent protein kinase (DNA-PK) cleavage (see Results), apoptosis was also assessed by quantitation of DEVDase activity against the Ac-DEVD-AMC substrate (CaspACE assay; Promega) and by a similar caspase 9 assay using Ac-LEHD-AMC (Enzyme Systems Products Inc.) as the substrate.

    Techniques: Expressing, Activity Assay, Western Blot

    PAT-SM6 mediates cytotoxicity to patient MM cells and MM cell lines by induction of apoptosis. A, MM cell lines were incubated with various concentrations of PAT-SM6 or control (unrelated IgM at highest concentration) for 72 h. Cell death was determined by FACS (Propidium iodide/LDS75 double positive cells). B, CD138-purified primary MM cells at primary diagnosis (n = 8, dots) or relapse (n = 3, rectangle) were incubated for 48 h with PAT-SM6 or isotype in media containing IL-6 and 10% FCS. Dead cells were determined by FACS analysis (Propidium iodide/LDS75 double positive cells). As control, PBMCs from healthy volunteers were treated under the same conditions. In contrast to controls PAT-SM6 significantly induced programmed cell death. C, MM1.S cells were incubated with PAT-SM6 or controls (staurophorin as positive control) for 72 h. AnnexinV and 7AAD positive cells were assessed by flow cytometry. Percentage of 7-AAD/Annexin V double positive cells, Isotype: 14%, PAT-SM6∶79% and positive control: 76%. D, MM1.S cells were incubated with PAT-SM6 or controls (staurophorin or isotype) for 6 h and caspase 3 activation was measured using the AC-DEVD-AMC protease assay. E, Bone marrow cells from a MM patient were CD33-purified to obtain myeloid progenitors and stimulated with PAT-SM6 or controls. No specific binding and subsequently no specific cytotoxicity was observed. F, Cells from the same patient as in E were CD138-isolated and incubated with PAT-SM6 (200 µg/mL) for 24 h. Specific PAT-SM6 binding and killing was observed.

    Journal: PLoS ONE

    Article Title: The Natural Human IgM Antibody PAT-SM6 Induces Apoptosis in Primary Human Multiple Myeloma Cells by Targeting Heat Shock Protein GRP78

    doi: 10.1371/journal.pone.0063414

    Figure Lengend Snippet: PAT-SM6 mediates cytotoxicity to patient MM cells and MM cell lines by induction of apoptosis. A, MM cell lines were incubated with various concentrations of PAT-SM6 or control (unrelated IgM at highest concentration) for 72 h. Cell death was determined by FACS (Propidium iodide/LDS75 double positive cells). B, CD138-purified primary MM cells at primary diagnosis (n = 8, dots) or relapse (n = 3, rectangle) were incubated for 48 h with PAT-SM6 or isotype in media containing IL-6 and 10% FCS. Dead cells were determined by FACS analysis (Propidium iodide/LDS75 double positive cells). As control, PBMCs from healthy volunteers were treated under the same conditions. In contrast to controls PAT-SM6 significantly induced programmed cell death. C, MM1.S cells were incubated with PAT-SM6 or controls (staurophorin as positive control) for 72 h. AnnexinV and 7AAD positive cells were assessed by flow cytometry. Percentage of 7-AAD/Annexin V double positive cells, Isotype: 14%, PAT-SM6∶79% and positive control: 76%. D, MM1.S cells were incubated with PAT-SM6 or controls (staurophorin or isotype) for 6 h and caspase 3 activation was measured using the AC-DEVD-AMC protease assay. E, Bone marrow cells from a MM patient were CD33-purified to obtain myeloid progenitors and stimulated with PAT-SM6 or controls. No specific binding and subsequently no specific cytotoxicity was observed. F, Cells from the same patient as in E were CD138-isolated and incubated with PAT-SM6 (200 µg/mL) for 24 h. Specific PAT-SM6 binding and killing was observed.

    Article Snippet: Caspase 3 Activation Assay using PAT-SM6 in the MM1.S Cell Line Determination of caspase 3 activation was performed using the AC-DEVD-AMC protease assay (BD Biosciences Pharmingen) as previously described by Mashima et al. .

    Techniques: Incubation, Concentration Assay, FACS, Purification, Positive Control, Flow Cytometry, Cytometry, Activation Assay, Protease Assay, Binding Assay, Isolation

    In vitro inhibition of caspase-3 activity by AMVIAP. Human caspase-9 activity was assayed in vitro with Ac-DEVD-AMC as the substrate. Caspase-3 was mixed with either, in the absence of recombinant IAPs (▪), GST at 2 μM (•), GST-XIAP at 4.1 μg (500 nM final) (+), or GST-AMVIAP at 2.9 μg (500 nM) (○). Activity was estimated by DEVD-AMC cleavage. Each data series represents fluorescence emission (490 nm) at 1-min intervals for 120 min.

    Journal: Journal of Virology

    Article Title: Functional Analysis of the Inhibitor of Apoptosis (iap) Gene Carried by the Entomopoxvirus of Amsacta moorei

    doi: 10.1128/JVI.79.4.2335-2345.2005

    Figure Lengend Snippet: In vitro inhibition of caspase-3 activity by AMVIAP. Human caspase-9 activity was assayed in vitro with Ac-DEVD-AMC as the substrate. Caspase-3 was mixed with either, in the absence of recombinant IAPs (▪), GST at 2 μM (•), GST-XIAP at 4.1 μg (500 nM final) (+), or GST-AMVIAP at 2.9 μg (500 nM) (○). Activity was estimated by DEVD-AMC cleavage. Each data series represents fluorescence emission (490 nm) at 1-min intervals for 120 min.

    Article Snippet: In a 96-well black plate, 50-μl protein samples were then mixed with 150 μl of reaction buffer (100 mM HEPES [pH 7.5], 10% sucrose, 0.1% CHAPS, 10 mM DTT) containing 14 μM fluorogenic tetrapeptide substrate Ac-DEVD-AMC [acetyl-Asp-Val-Ala-Asp-(amino-4-methyl coumarin)] (Sigma).

    Techniques: In Vitro, Inhibition, Activity Assay, Recombinant, Fluorescence

    Rescue of cells from hid -induced apoptosis by p35 and iap genes. Transfection with hid induces apoptosis and prevents expression of GFP from a second cotransfected plasmid. (A) The rescue of hid -induced apoptosis in Ld652 cells was shown as the rescue of GFP expression in cells after transfection with mock (a), (b) vector-vector (b), hid- vector (c), hid-p35 (1:1) (d), hid- Op iap (1:1) (e), hid- AMV iap (1:1) (f), hid- p35 (1:8) (g), hid- Op iap (1:8) (h), hid- AMV iap (1:8) (i). All samples were also cotransfected with 2 μg of the indicator pIE1 gfp , 0.5 μg of pIE1 hid , and either 4 μg of pIE1 vector or pIE1 plasmids containing apoptotic suppressor genes at the ratio indicated for each transfection. Photos were taken at 48 h after transfection in a Zeiss inverted phase-contrast microscope (magnification, ×20). (B) Caspase-3-like activity in Ld652 cells as measured by Ac-DEVD-AMC cleavage. Cells were cotransfected with 2 μg of pIE gfp as the indicator plasmid, 0.5 μg of pIE1 hid , and a third plasmid, either pIE1-4 vector or pIE1-containing one of the apoptotic suppressor genes ( p35 , Op- iap , or AMV iap ) at different ratios of hid to the third plasmid, ranging from 1:1 up to 1:32. Relative value units of caspase activity were expressed as the FSU per second in 5 × 10 4 cells.

    Journal: Journal of Virology

    Article Title: Functional Analysis of the Inhibitor of Apoptosis (iap) Gene Carried by the Entomopoxvirus of Amsacta moorei

    doi: 10.1128/JVI.79.4.2335-2345.2005

    Figure Lengend Snippet: Rescue of cells from hid -induced apoptosis by p35 and iap genes. Transfection with hid induces apoptosis and prevents expression of GFP from a second cotransfected plasmid. (A) The rescue of hid -induced apoptosis in Ld652 cells was shown as the rescue of GFP expression in cells after transfection with mock (a), (b) vector-vector (b), hid- vector (c), hid-p35 (1:1) (d), hid- Op iap (1:1) (e), hid- AMV iap (1:1) (f), hid- p35 (1:8) (g), hid- Op iap (1:8) (h), hid- AMV iap (1:8) (i). All samples were also cotransfected with 2 μg of the indicator pIE1 gfp , 0.5 μg of pIE1 hid , and either 4 μg of pIE1 vector or pIE1 plasmids containing apoptotic suppressor genes at the ratio indicated for each transfection. Photos were taken at 48 h after transfection in a Zeiss inverted phase-contrast microscope (magnification, ×20). (B) Caspase-3-like activity in Ld652 cells as measured by Ac-DEVD-AMC cleavage. Cells were cotransfected with 2 μg of pIE gfp as the indicator plasmid, 0.5 μg of pIE1 hid , and a third plasmid, either pIE1-4 vector or pIE1-containing one of the apoptotic suppressor genes ( p35 , Op- iap , or AMV iap ) at different ratios of hid to the third plasmid, ranging from 1:1 up to 1:32. Relative value units of caspase activity were expressed as the FSU per second in 5 × 10 4 cells.

    Article Snippet: In a 96-well black plate, 50-μl protein samples were then mixed with 150 μl of reaction buffer (100 mM HEPES [pH 7.5], 10% sucrose, 0.1% CHAPS, 10 mM DTT) containing 14 μM fluorogenic tetrapeptide substrate Ac-DEVD-AMC [acetyl-Asp-Val-Ala-Asp-(amino-4-methyl coumarin)] (Sigma).

    Techniques: Transfection, Expressing, Plasmid Preparation, Microscopy, Activity Assay

    Induction of caspase-3-like activity in cell lines infected with vAmΔ sph / gfp or vAm gfp /Δ iap/lacZ . Caspase-3-like activity was examined in protein samples comprising combined supernatants and cell pellets at the times indicated. Caspase-3-like activity was measured by the cleavage of Ac-DEVD-AMC. S2 (A), Sf9 (B), and Ld652 (C) cells were infected with vAmΔ sph / gfp or vAm gfp /Δ iap/lacZ at an MOI of 15.

    Journal: Journal of Virology

    Article Title: Functional Analysis of the Inhibitor of Apoptosis (iap) Gene Carried by the Entomopoxvirus of Amsacta moorei

    doi: 10.1128/JVI.79.4.2335-2345.2005

    Figure Lengend Snippet: Induction of caspase-3-like activity in cell lines infected with vAmΔ sph / gfp or vAm gfp /Δ iap/lacZ . Caspase-3-like activity was examined in protein samples comprising combined supernatants and cell pellets at the times indicated. Caspase-3-like activity was measured by the cleavage of Ac-DEVD-AMC. S2 (A), Sf9 (B), and Ld652 (C) cells were infected with vAmΔ sph / gfp or vAm gfp /Δ iap/lacZ at an MOI of 15.

    Article Snippet: In a 96-well black plate, 50-μl protein samples were then mixed with 150 μl of reaction buffer (100 mM HEPES [pH 7.5], 10% sucrose, 0.1% CHAPS, 10 mM DTT) containing 14 μM fluorogenic tetrapeptide substrate Ac-DEVD-AMC [acetyl-Asp-Val-Ala-Asp-(amino-4-methyl coumarin)] (Sigma).

    Techniques: Activity Assay, Infection

    Loperamide, pimozide- or STF-62247-induced cell death does not primarily involve apoptosis, ferroptosis, or necroptosis. a , c , d MZ-54 cells were pretreated for 1 h with 20 µM zVAD.fmk ( a ), 5 µM Fer-1 ( c ) or 30 µM Nec-1s ( d ) followed by treatment with 17.5 µM loperamide, 15 µM pimozide, 40 µM STF-62247, 20 µM IM/100 µM TIC, 25 µM ABT-737/100 µM etoposide, 500 nM RSL3 or 1 ng/mL TNF \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\alpha}$$\end{document} α + 0.5 µM BV6 for 48 h. Cell death was assessed by measuring the PI uptake as fraction of total nuclei determined by Hoechst counterstaining using high-content fluorescence microscopy. HT-29 cells served as positive control for induction of necroptotic cell death. b MZ-54 cells were treated with 3 µM STS, 15 µM loperamide, 15 µM pimozide, 40 µM STF-62247, or 20 µM IM/100 µM TIC for the indicated time points. Caspase-3 activity was determined by quantifying alterations in Ac-DEVD-AMC fluorescence. Mean and SEM of 3−4 independent experiments performed in triplicate are shown. * p

    Journal: Cell Death & Disease

    Article Title: Loperamide, pimozide, and STF-62247 trigger autophagy-dependent cell death in glioblastoma cells

    doi: 10.1038/s41419-018-1003-1

    Figure Lengend Snippet: Loperamide, pimozide- or STF-62247-induced cell death does not primarily involve apoptosis, ferroptosis, or necroptosis. a , c , d MZ-54 cells were pretreated for 1 h with 20 µM zVAD.fmk ( a ), 5 µM Fer-1 ( c ) or 30 µM Nec-1s ( d ) followed by treatment with 17.5 µM loperamide, 15 µM pimozide, 40 µM STF-62247, 20 µM IM/100 µM TIC, 25 µM ABT-737/100 µM etoposide, 500 nM RSL3 or 1 ng/mL TNF \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\alpha}$$\end{document} α + 0.5 µM BV6 for 48 h. Cell death was assessed by measuring the PI uptake as fraction of total nuclei determined by Hoechst counterstaining using high-content fluorescence microscopy. HT-29 cells served as positive control for induction of necroptotic cell death. b MZ-54 cells were treated with 3 µM STS, 15 µM loperamide, 15 µM pimozide, 40 µM STF-62247, or 20 µM IM/100 µM TIC for the indicated time points. Caspase-3 activity was determined by quantifying alterations in Ac-DEVD-AMC fluorescence. Mean and SEM of 3−4 independent experiments performed in triplicate are shown. * p

    Article Snippet: 50 μl of cell lysate were added to 150 µL reaction buffer (25 mM HEPES, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, 3 mM DTT, pH 7.5) containing the fluorigenic substrate Ac-DEVD-AMC (Enzo Life Sciences, Lausen, Switzerland) at a final concentration of 10 μM.

    Techniques: Fluorescence, Microscopy, Positive Control, Activity Assay

    Cell proliferation and caspase-3 activity assay. (A) Stable overexpression of CYP2E1 inhibits the cell growth of HepG2 cells. E47 and C34 cells, two stably transfected HepG2 cell lines harboring CYP2E1 recombinant gene or its empty vector respectively, were seeded with the same number in triplicate on 96-well plates and cultured for up to 6 days. Each day, the triplicate of cultured cells was harvested and subjected to CCK-8 assay at 450 nm. The amount of the formazan dye, generated by the activities of dehydrogenases in cells, is directly proportional to the number of living cells. (B) Activities of caspase-3 increased in HepG2 cells which stably expresses ectopic CYP2E1. E47 and C34 cells were seeded in triplicate on 96-well plates. Cells were harvested on day 1, 3 and 6, and cell lysates were used to test the activities of caspase-3 by measuring proteolytic cleavage of the proluminescent substrates AC-DEVD-AMC. The fluorescence was detected to reflect the amount of released AMC (caspase-3, λex = 380, λem = 460). The results were expressed as arbitrary units of fluorescence (AUF) per milligram of lysate protein.

    Journal: PLoS ONE

    Article Title: HBx Inhibits CYP2E1 Gene Expression via Downregulating HNF4α in Human Hepatoma Cells

    doi: 10.1371/journal.pone.0107913

    Figure Lengend Snippet: Cell proliferation and caspase-3 activity assay. (A) Stable overexpression of CYP2E1 inhibits the cell growth of HepG2 cells. E47 and C34 cells, two stably transfected HepG2 cell lines harboring CYP2E1 recombinant gene or its empty vector respectively, were seeded with the same number in triplicate on 96-well plates and cultured for up to 6 days. Each day, the triplicate of cultured cells was harvested and subjected to CCK-8 assay at 450 nm. The amount of the formazan dye, generated by the activities of dehydrogenases in cells, is directly proportional to the number of living cells. (B) Activities of caspase-3 increased in HepG2 cells which stably expresses ectopic CYP2E1. E47 and C34 cells were seeded in triplicate on 96-well plates. Cells were harvested on day 1, 3 and 6, and cell lysates were used to test the activities of caspase-3 by measuring proteolytic cleavage of the proluminescent substrates AC-DEVD-AMC. The fluorescence was detected to reflect the amount of released AMC (caspase-3, λex = 380, λem = 460). The results were expressed as arbitrary units of fluorescence (AUF) per milligram of lysate protein.

    Article Snippet: Caspase-3 activity assay in C34 and E47 cells Caspase-3 activities were determined in cell lysate by measuring proteolytic cleavage of the proluminescent substrates AC-DEVD-AMC (Calbiochem, La Jolla, CA).

    Techniques: Caspase-3 Activity Assay, Over Expression, Stable Transfection, Transfection, Recombinant, Plasmid Preparation, Cell Culture, CCK-8 Assay, Generated, Fluorescence

    Op-IAP 1–216 acceleration of caspase activation. SF21 cells were UV irradiated 18 h after transfection with a plasmid encoding Op-IAP HA/1–216 (solid bars) or the vector alone (striped bars). Cell extracts prepared immediately prior to irradiation (0 h) or at the indicated times after irradiation were assayed for caspase activity by using Ac-DEVD-AMC as the substrate. Relative caspase activities are averages ± the standard deviations of triplicate assays. Caspase activity in extracts from stably transfected Op-IAP cell line E6 after UV irradiation (open bars) was included.

    Journal: Molecular and Cellular Biology

    Article Title: The BIR Motifs Mediate Dominant Interference and Oligomerization of Inhibitor of Apoptosis Op-IAP

    doi:

    Figure Lengend Snippet: Op-IAP 1–216 acceleration of caspase activation. SF21 cells were UV irradiated 18 h after transfection with a plasmid encoding Op-IAP HA/1–216 (solid bars) or the vector alone (striped bars). Cell extracts prepared immediately prior to irradiation (0 h) or at the indicated times after irradiation were assayed for caspase activity by using Ac-DEVD-AMC as the substrate. Relative caspase activities are averages ± the standard deviations of triplicate assays. Caspase activity in extracts from stably transfected Op-IAP cell line E6 after UV irradiation (open bars) was included.

    Article Snippet: After clarification by centrifugation (16,000 × g ), caspase activity was determined by using 10 μM Ac-DEVD-AMC (Peptides International) as the substrate in 100-μl reaction mixtures with protease buffer (25 mM HEPES [pH 7.5], 0.1% CHAPS, 10% sucrose, 1 mM EDTA, 10 mM dithiothreitol) and 0.005% bovine serum albumin.

    Techniques: Activation Assay, Irradiation, Transfection, Plasmid Preparation, Activity Assay, Stable Transfection

    ( A – J ) Biocompatibility of C–Co–SiO 2 and C–Co 3 O 4 –SiO 2 with lung respiration, adenosine triphosphate (ATP) content, caspase activity, and histology. ( A and B ) Lung specimens were incubated in vitro at 37°C in oxygenated Krebs–Henseleit (KH) buffer with and without 0.2 mg/dL C–Co–SiO 2 or C–Co 3 O 4 –SiO 2 . A summary of the values of k c (μM O 2 /minute/mg) for all experiments is shown in ( A ). The P -values are for comparisons with the untreated condition; “n” is the number of independent measurements. Representative high-performance liquid chromatography (HPLC) runs at 4 hours are shown in ( B ). Briefly, lung specimens were incubated at 37°C with 37 Ac-DEVD-AMC μM for 20 minutes. The tissues were then disrupted by vigorous homogenization, and the supernatants were separated on HPLC and analyzed for the free AMC moiety. Ac-DEVD-AMC had a retention time, R t , of ~4.5 minutes (insert) and AMC of ~12.7 minutes (running solvents, CH 3 CN:dH 2 O 1:3). ( C – F ) Lung specimens were collected 60 minutes after intratracheal instillation of 10 mg C–Co–SiO 2 , 10 mg C–Co 3 O 4 –SiO 2 , or 150 μL 0.9% NaCl. The samples were then incubated in vitro at 37°C in oxygenated KH buffer. Cellular respiration and caspase activity were measured as a function of time. Representative O 2 runs are show in ( C and D ); the values of k c (μM O 2 /minute/mg) are shown at the bottom of each run. Representative HPLC runs of the samples 4 hours after incubation of the specimen with the nanoparticles are shown in ( E ). Ac-DEVD-AMC had a retention time, R t , of ~2.5 minutes (insert) and AMC of ~4.7 minutes (running solvents, methanol:dH 2 O 1:1). The AMC peak area is plotted as a function of time in ( F ). ( G – J ) Representative histology (hematoxylin and eosin) 4 hours after in vitro treatment ( G and H ) and intratracheally treated ( I and J ) lungs with C–Co–SiO 2 ( G and I ), or C–Co 3 O 4 –SiO 2 ( H and J ). The in vitro treatment with C–Co–SiO 2 ( G , 40×) revealed necrosis of the alveolar wall (arrow) and numerous apoptotic bodies (arrowheads), compared with a better-preserved area of the alveolar wall. Similar alterations were noted with C–Co 3 O 4 –SiO 2 ( H , 20×). The in vivo treatment with C–Co–SiO 2 revealed focal areas of alveolar wall damage and apoptotic bodies (arrows) adjacent to bronchioles. ( I , 20×). The in vivo treatment with C–Co 3 O 4 –SiO 2 revealed pulmonary tissue with preserved architecture. The alveolar wall is thinned, and there is vacuolization of pneumocytes and endothelial cells, with loss of definite cell outlines ( J , 20×).

    Journal: International Journal of Nanomedicine

    Article Title: Lung toxicities of core-shell nanoparticles composed of carbon, cobalt, and silica

    doi: 10.2147/IJN.S39649

    Figure Lengend Snippet: ( A – J ) Biocompatibility of C–Co–SiO 2 and C–Co 3 O 4 –SiO 2 with lung respiration, adenosine triphosphate (ATP) content, caspase activity, and histology. ( A and B ) Lung specimens were incubated in vitro at 37°C in oxygenated Krebs–Henseleit (KH) buffer with and without 0.2 mg/dL C–Co–SiO 2 or C–Co 3 O 4 –SiO 2 . A summary of the values of k c (μM O 2 /minute/mg) for all experiments is shown in ( A ). The P -values are for comparisons with the untreated condition; “n” is the number of independent measurements. Representative high-performance liquid chromatography (HPLC) runs at 4 hours are shown in ( B ). Briefly, lung specimens were incubated at 37°C with 37 Ac-DEVD-AMC μM for 20 minutes. The tissues were then disrupted by vigorous homogenization, and the supernatants were separated on HPLC and analyzed for the free AMC moiety. Ac-DEVD-AMC had a retention time, R t , of ~4.5 minutes (insert) and AMC of ~12.7 minutes (running solvents, CH 3 CN:dH 2 O 1:3). ( C – F ) Lung specimens were collected 60 minutes after intratracheal instillation of 10 mg C–Co–SiO 2 , 10 mg C–Co 3 O 4 –SiO 2 , or 150 μL 0.9% NaCl. The samples were then incubated in vitro at 37°C in oxygenated KH buffer. Cellular respiration and caspase activity were measured as a function of time. Representative O 2 runs are show in ( C and D ); the values of k c (μM O 2 /minute/mg) are shown at the bottom of each run. Representative HPLC runs of the samples 4 hours after incubation of the specimen with the nanoparticles are shown in ( E ). Ac-DEVD-AMC had a retention time, R t , of ~2.5 minutes (insert) and AMC of ~4.7 minutes (running solvents, methanol:dH 2 O 1:1). The AMC peak area is plotted as a function of time in ( F ). ( G – J ) Representative histology (hematoxylin and eosin) 4 hours after in vitro treatment ( G and H ) and intratracheally treated ( I and J ) lungs with C–Co–SiO 2 ( G and I ), or C–Co 3 O 4 –SiO 2 ( H and J ). The in vitro treatment with C–Co–SiO 2 ( G , 40×) revealed necrosis of the alveolar wall (arrow) and numerous apoptotic bodies (arrowheads), compared with a better-preserved area of the alveolar wall. Similar alterations were noted with C–Co 3 O 4 –SiO 2 ( H , 20×). The in vivo treatment with C–Co–SiO 2 revealed focal areas of alveolar wall damage and apoptotic bodies (arrows) adjacent to bronchioles. ( I , 20×). The in vivo treatment with C–Co 3 O 4 –SiO 2 revealed pulmonary tissue with preserved architecture. The alveolar wall is thinned, and there is vacuolization of pneumocytes and endothelial cells, with loss of definite cell outlines ( J , 20×).

    Article Snippet: Ac-DEVD-AMC (MW 729.6) was obtained from Axxora (San Diego, CA, USA).

    Techniques: Activity Assay, Incubation, In Vitro, High Performance Liquid Chromatography, Homogenization, In Vivo

    D609 enhances FasL-induced caspase-independent ceramide increase and cell death. Jurkat cells (clone A3) were pre-incubated in the presence or absence of 40 μM zVAD-fmk for 1 h and further incubated with or without FasL (500 ng/mL) for 16 h or the indicated times. Ceramide level (expressed as nmol of ceramide per mg of protein) ( A ) and caspase activity toward Ac-DEVD-AMC ( B ) were measured. ( C ) Cells were pre-incubated for 1 h with 40 μM zVAD-fmk (black bars) or a combination of 40 μM zVAD-fmk and 50 μg/mL D609 (white bars). Cells were further incubated with 500 ng/mL FasL for the indicated times and ceramide concentration was measured. Data are expressed as the percentage of values measured in cells incubated with zVAD-fmk alone. ( A – C ) Values are means ± S.E.M. of three independent experiments. ( D ) Cells were pre-incubated for 1 h with 40 μM zVAD-fmk in the presence (triangles) or absence (squares) of 50 μg/mL D609. Cells were further incubated for the indicated times with (solid symbols) or without (empty symbols) 500 ng/mL FasL. Cell death was evaluated by flow cytometry after annexin-V-FITC and propidium iodide labeling. Under these conditions, most of the dead cells were labeled by both annexin-V-FITC and propidium iodide. Data are representative of two independent experiments.

    Journal: International Journal of Molecular Sciences

    Article Title: The Tricyclodecan-9-yl-xanthogenate D609 Triggers Ceramide Increase and Enhances FasL-Induced Caspase-Dependent and -Independent Cell Death in T Lymphocytes

    doi: 10.3390/ijms13078834

    Figure Lengend Snippet: D609 enhances FasL-induced caspase-independent ceramide increase and cell death. Jurkat cells (clone A3) were pre-incubated in the presence or absence of 40 μM zVAD-fmk for 1 h and further incubated with or without FasL (500 ng/mL) for 16 h or the indicated times. Ceramide level (expressed as nmol of ceramide per mg of protein) ( A ) and caspase activity toward Ac-DEVD-AMC ( B ) were measured. ( C ) Cells were pre-incubated for 1 h with 40 μM zVAD-fmk (black bars) or a combination of 40 μM zVAD-fmk and 50 μg/mL D609 (white bars). Cells were further incubated with 500 ng/mL FasL for the indicated times and ceramide concentration was measured. Data are expressed as the percentage of values measured in cells incubated with zVAD-fmk alone. ( A – C ) Values are means ± S.E.M. of three independent experiments. ( D ) Cells were pre-incubated for 1 h with 40 μM zVAD-fmk in the presence (triangles) or absence (squares) of 50 μg/mL D609. Cells were further incubated for the indicated times with (solid symbols) or without (empty symbols) 500 ng/mL FasL. Cell death was evaluated by flow cytometry after annexin-V-FITC and propidium iodide labeling. Under these conditions, most of the dead cells were labeled by both annexin-V-FITC and propidium iodide. Data are representative of two independent experiments.

    Article Snippet: Fluorogenic DEVD Cleavage Enzyme Assays After incubation with FasL, cells were sedimented and caspase-like activities were assessed using Ac-DEVD-AMC (Bachem) as described elsewhere [ ].

    Techniques: Incubation, Activity Assay, Concentration Assay, Flow Cytometry, Cytometry, Labeling