recombinant caspase-3 Search Results


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  • 99
    Thermo Fisher recombinant caspase 3
    ( A ) Ligation of (1–45)αCOSR and (46–142) at 1.5 h. The reaction was monitored by analytical HPLC on a Waters XBridge C 18 column (4.6 × 150 mm, 3.5 μM) running a 30-min gradient of 25%–45% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. ( Insets ) Mass spectra of (1–45)αCOSR and (46–142) determined by ESI-MS. ( B ) Ligated full-length survivin characterized by C 18 RP-HPLC and ESI-MS. HPLC conditions: Waters symmetry 300 C 18 column (4.6 × 150 mm, 5 μM) running a 30-min gradient of 5%–65% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. ( C ) CD spectra of synthetic survivin at 5 μM in 5 mM phosphate buffer containing 0.1 mM TCEP, pH 7.5 (thin line), and at 25 μM in 5 mM phosphate buffer containing 0.1 mM TCEP and 50 μM Zn 2+ , pH 7.5 (thick line). ( D ) Representative size-exclusion chromatograms of synthetic survivin (3) and molecular mass standards (1, 2, 4, 5). Linear regression analysis of the correlation between logarithmic M r and retention time is illustrated in the inset . ( E ) Representative raw data from the hydrolysis of Ac-DEVD-AMC by <t>caspase-3</t> in the absence and presence of different concentrations of XIAP and synthetic survivin. ( F ) Dose-dependent percent inhibition of caspase-3 by XIAP (filled circles), synthetic survivin without Zn 2+ (empty squares), and synthetic survivin in the presence of Zn 2+ (filled squares). Each curve is the mean of three independent experiments.
    Recombinant Caspase 3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore human recombinant caspase 3
    Relative resistance of PrP C from mouse brain lysate to calpain proteolysis . Lysate from naïve mouse brains were prepared and digested with calpain-1, -2, or <t>caspase-3</t> as described in section “Materials and Methods.” Aliquots of the untreated and treated lysates were then electrophoresed, western blotted, and immunostained with either anti-αII-spectrin Ab (A) or with anti-PrP Mab E11. (B) Characteristic calpain-generated SBDP150 and SBDP145 as well as caspase-3 generated SBDP150i and SBDP120 are indicated. No PrP C -BDPs were observed following the treatments with Mab 7E4 (data not shown).
    Human Recombinant Caspase 3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Upstate Biotechnology Inc recombinant caspase 3
    Examination of caspase activity in cocultures incubated under simulated ischemia or elevated hydrostatic pressure. A , Western blot analysis of caspase-8 expression in cocultures. B , Western blot analysis of <t>caspase-3</t> expression cocultures. Column 1 , Control retinal ganglion cells; column 2 , retinal ganglion cells incubated under simulated ischemia for 72 hr; column 3 , retinal ganglion cells incubated under elevated hydrostatic pressure for 72 hr; column 4 , control glial cells; column 5 , glial cells incubated under simulated ischemia for 72 hr; column 6 , glial cells incubated under elevated hydrostatic pressure for 72 hr. Western blots revealed that the 55 kDa immunoreactive band corresponding to caspase-8 cleaved to 30 and 20 kDa products in retinal ganglion cells that were incubated under stress conditions. In addition, 32 kDa pro-enzyme caspase-3 cleaved to a 17 kDa active subunit in retinal ganglion cells. No cleavage of caspase-8 or caspase-3 was detected with the use of the extracts of glial cells. Caspase activation also was examined, in situ , using Phiphilux-G 6 D 2 in retinal ganglion cells that were incubated under different conditions for 72 hr. C , Normal conditions. D , Simulated ischemia. E , Elevated hydrostatic pressure. Fluorescence microscope images seen in F–H correspond to phase-contrast images of the retinal ganglion cells seen in C–E , respectively. Rhodamine fluorescence ( red ) indicates caspase-3-like activity in retinal ganglion cells that were incubated under stress conditions. I , The amount of DEVD-AMC cleaving activity with the use of fluorometric analysis was higher in retinal ganglion cells in cocultures that were incubated under simulated ischemia or elevated hydrostatic pressure for 72 hr as compared with cocultures that were incubated under normal conditions (Mann–Whitney U test; p = 0.006 and p = 0.04, respectively).
    Recombinant Caspase 3, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 81/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Enzo Biochem recombinant caspase 3
    GRASP65 cleavage is an early event during apoptosis. ( a ) Time-lapse imaging of GRASP65–GFP caspase cleavage. To the top, phase contrast images of a TRAIL-treated HeLa cell before and after the onset of apoptosis; to the bottom, fluorescence images of Golgi-associated GRASP65–GFP and the loss of GFP signal from the Golgi following the onset of apoptosis. ( b ) Normalised fluorescence intensity traces of Golgi-associated GRASP65–GFP in HeLa cells treated with the apoptosis-inducing reagents shown. Traces are plotted relative to the onset of cell rounding (hatched boxes). Data show means ± S.D. for a minimum of five cells per treatment. ( c ) Autoradiographs of 35 S-labelled GRASP65 translated in vitro and incubated for 90 min with increasing concentrations of recombinant <t>caspase-3</t> or caspase-8. Full-length (
    Recombinant Caspase 3, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 83/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore recombinant caspase 3
    Caspase cleavage of pBim EL in T cells at an early stage of apoptosis. ( A ) Caspase cleavage of pBim EL in apoptotic Jurkat T cells. Anti-Bim Western blotting was performed with the rabbit polyclonal antibodies directed against the full-length Bim EL to examine the purified Flag-tagged rBim EL and Flag-tagged rBim L (lanes 1 and 2), Jurkat cell lysates incubated with (lane 5) or without (lanes 3 and 4) PAP, and lysates from Jurkat cells subjected to the various treatments as indicated (lanes 7–20). In lanes 19 and 20, the antibody solution was preincubated with purified rBim EL before being used in Western blotting. In lanes 21–24, Western blotting was performed with anti-Bim antibodies obtained from StressGen Biotechnologies (Victoria, Canada) and ProMab Biotechnologies (Mountain View, CA), respectively. ( B ) Cleavage of pBim EL at an early stage of apoptosis. Lysates of Jurkat cells treated with staurosporine for the indicated periods of time (hr) were analyzed by anti-Bim Western blotting. The percentage of apoptotic cells was determined by flow-cytometric analysis and is indicated at the bottom. ( C ) Apoptosis-induced cleavage of pBim EL in activated mouse primary T cells. Anti-Bim Western blot analysis of lysates from activated mouse primary T cells treated with or without staurosporine. ( D ) pBim EL cleaved in vitro by recombinant caspases-3 comigrates with tBim EL from apoptotic Jurkat cells as indicated by anti-Bim Western blotting. Lanes 1 and 2, lysates from healthy Jurkat cells were incubated with or without recombinant <t>caspase-3.</t> Lanes 3 and 4 show lysates from Jurkat cells treated with or without staurosporine. Stau, staurosporine; OA, okadaic acid; mBim EL , modified Bim EL .
    Recombinant Caspase 3, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Pharmingen recombinant caspase 3
    Requirement for an intact BH3 domain on the cytoplasmic face of the mitochondrial outer membrane. ( A ) Sequence of p15 BID BH3 domain mutants mIII.1 and mIII.4. Shaded box indicates the <t>caspase-3</t> cleavage site DEMD that generates the p11 fragment. ( B ) Treatment of mitochondria with recombinant caspase-3 after targeting wild-type (wt) and caspase-cleavage site mutant p15 BID mIII.1. (Lane 1 ) Amount of p15 BID targeted. (Lane 2 ) Treatment with caspase-3 at 4°C. (Lane 3 ) Treatment with caspase-3 at 30°C. p15 BID and p11 BID are visualized by 35 S fluorography. ( C ) In vitro targeting and cytochrome c release by p15 BID wild type vs mIII.4. (*) A cross-reactive protein present in IVT mix. ( D ) In vitro targeting and cytochrome c release by BIDα345/TM wild-type and mutant mIII.4.
    Recombinant Caspase 3, supplied by Pharmingen, used in various techniques. Bioz Stars score: 81/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Sino Biological recombinant caspase 3
    Western blot was utilized to evaluate the levels of <t>pro-caspase-3,</t> cleaved caspase-3 and α -tubulin. ( a ) SFN-Cys downregulated the level of pro-caspase-3. ( b ) SFN-Cys upregulated the expression of cleaved caspase-3 via increased concentrations; ( c ) and ( d ) SFN-Cys decreased α -tubulin, whereas PD98059 reserved this process. ( e ) In vitro , recombinant caspase-3 cleaved α -tubulin to produce a band of 53 kDa, suggesting that SFN-Cys activated caspase-3 might degrade α -tubulin. Each experiment was performed in triplicate. Error bars indicate uncertainty errors in graphs to relay 95% of confidence in the interpreted data. * Indicates P
    Recombinant Caspase 3, supplied by Sino Biological, used in various techniques. Bioz Stars score: 88/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Biomol GmbH recombinant caspase 3
    Calpain activation in acute and chronic 3-NP models. A–C , Homogenates prepared from striatum and cerebral cortex of 3-NP-treated and control (CTRL) rats were analyzed by SDS-PAGE followed by Western blot for fodrin (known substrate of calpain and <t>caspase-3)</t> and assayed for calpain-dependent proteolytic activity using the fluorogenic substrate N -succinyl-LY-AMC ( D ). E , Western blot analysis ofμ-calpain cleavage in cytosol fractions from striatum and cerebral cortex of 3-NP-treated animals. The typical calpain-dependent cleavage of fodrin appearing as a 145/150 kDa doublet is found in the striatum of both acute ( A ) and chronic ( B ) 3-NP models (each lane contains pooled samples from 5–8 animals). Note in A the presence between two nonspecific (N.S.) bands of a 120 kDa band (asterisk), possibly because of a caspase-3-mediated fodrin breakdown product. In B , chronic 3-NP animals were analyzed every 6 hr showing that the intensity of this doublet increases on day 4 and is maximal at day 5. Western blots for fodrin in cerebral cortex were slightly overexposed. C shows that the pattern found in the striatum of 3-NP-treated animals (day 5) is clearly different from the caspase-3-mediated cleavage of fodrin observed after in vitro digestion of control samples with recombinant caspase-3 (CTRL + R. Casp-3). Note the prominent increase in the proteolytic activity of calpain and the presence of cleaved/active form ofμ-calpain in the striatum of chronic 3-NP rats, whereas no changes were found within the cerebral cortex of the same animals. Data are means ± SEM determined in 5–10 animals. N.D., Not detectable. ★ p
    Recombinant Caspase 3, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 81/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    BIO-CAT recombinant caspase 3
    Spectral response and caspase sensing of CaspeR3 . A. Excitation (dashed lines) and emission (solid lines) spectra for TagGFP (green) and TagRFP (red). Spectral overlap is filled with gray. B. Emission spectra of CaspeR3 before (dashed line) and after digestion by <t>Caspase</t> 3 (solid line). C. Green-to-red emission ratio change of CaspeR3 upon staurosporine-induced apoptosis. Approximately 40–50 min after staurosporine infusion, cells demonstrated pronounced changes fluorescence signal ratio. Emission ratio shown for 5 cells, time point aligned to the median of ratio changes, individual for each cell. Excitation at 488 nm, emission was detected at 500–530 nm and 560–600 nm. D. TagGFP fluorescence lifetime τ φ (solid lines) and τ M (dashed lines) changes for CaspeR3 during staurosporine-induced apoptosis. Excitation was at 488 nm and donor fluorescence emission was passed through a 500–530 nm bandpass filter. E. Two channel fluorescence imaging of CaspeR3 upon staurosporine-induced apoptosis in HeLa cells. Time, in minutes, is shown after staurosporine infusion.
    Recombinant Caspase 3, supplied by BIO-CAT, used in various techniques. Bioz Stars score: 80/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Becton Dickinson recombinant caspase 3
    δ-Catenin is cleaved by <t>caspase-3</t> in vitro and in apoptotic extracts. A , biotin-conjugated Xenopus δ-catenin was cleaved by recombinant active caspase-3 in vitro . Intact protein or cleaved fragments (labeled with asterisks ) were visualized
    Recombinant Caspase 3, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 80/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    R&D Systems recombinant caspase 3
    NS3694 inhibits TNF-induced effector caspase activation and apoptosis in <t>MCF-casp3</t> cells. MCF-casp3 cells were left untreated (UN) or pretreated with the indicated concentrations of DEVD-CHO or zVAD-fmk for 1 h before the indicated concentrations of TNF were added. NS3694 was added at 10 to 100 μM at the same time as TNF. (A) The survival of the cells was analyzed by the MTT assay after 48 h of TNF treatment. The means ± standard deviations (error bars) of three independent experiments are shown. (B) After 17 h of treatment, the cells were lysed, and the DEVDase activity in lysates was assessed by spectrofluorometric quantification of DEVD-AFC cleavage and presented as picomoles per minute. The means ± standard deviations (error bars) for three samples in a representative experiment are shown. The experiments were repeated several times with similar results.
    Recombinant Caspase 3, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    BioVision recombinant caspase 3
    Cleavage of BeAn virions in vitro by <t>caspase-3</t> produces 13- and 17-kDa peptides and an increased physical particle/PFU ratio.
    Recombinant Caspase 3, supplied by BioVision, used in various techniques. Bioz Stars score: 86/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam recombinant caspase 3
    SASH1 overexpression induces apoptosis. ( a ) HeLa cells were transfected with Flag-SASH1 (OE=overexpressed) and irradiated with ultraviolet light C (UVC) 3 h to induce apoptosis (10–50 mJ/cm 2 ). Quantification of PARP1, cleaved <t>caspase-3</t> and cleaved caspase-9 levels indicated below blots wasperformed with ImageJ (University of Wisconsin, Madison, USA). Statistical analysis was performed with Student's T -test with * P
    Recombinant Caspase 3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    Millipore active recombinant caspase 3
    Design and pharmacological evaluation of TRP601. ( a ) Substrate binding region of caspase-2 (Casp2) in complex with TRP601. The quinolin-2-carbonyl-Val-Asp(OMe)-Val-Ala-Asp(OMe)-CH 2 - moiety is shown in sticks representation with the atoms represented as follows: gray=carbon; white=hydrogen; blue=nitrogen; and red=oxygen. The S–C covalent bond between the Cys-155 residue and the C terminus CH 2 of TRP601 is represented in yellow. The electric interactions (hydrogen bonds and salts bridges) are represented by the dashed, green lines. The enzyme residues that interact with the inhibitor are shown by the wireframe representation. Right panel: the table shows the minimal energy (kcal/mol, E min ) of the Casp2–TRP601 complex resulting from electric or Coulombic component ( E c ) together with repulsion–attraction component ( E vw ). The interaction energy of the Casp2–TRP601 complex is −147.28 kcal/mol. Owing to the C–S covalent bond, the Asp residue in P1 contributes to about 30% of the interaction energy. The Asp in P4 and the Val in P5 have important non-covalent contributions to stabilization of the complex, 21% and 17%, respectively. Each of the other two residues (Ala in P2 and Val in P3) contributes to about 12% of the interaction energy. ( b ) Structure of TRP601. ( c ) Representative dose–response curve of human recombinant Casp2 and <t>Casp3</t> inhibition by TRP601. Initial velocities were determined from standard colorimetric microplate assays. ( d ) Kinetic off rate (k 3 / K i ) parameters of irreversible caspase inhibitors on Casp2 and Casp3. ( e ) TRP601 inhibits neuronal caspase activities and prevents serum deprivation (SD)-induced cell death. High-density E14 cortical neuron cultures were subjected to 24 h SD in the presence or absence of 50 μM TRP601. Histograms indicate the means (±S.D.) of 15 independent experiments. ( f ) Representative pharmacokinetic of TRP601 after intravenous (i.v.) administration in adult rats, through liquid chromatography-mass spectrometry (LC-MS/MS) detection in the plasma and brain homogenates. Note that following intraperitoneal (i.p.) administration of the same dose, TRP601 was detected in the brain at 0.25 h (brain C max =25 ng/ml) and the C max (20 ng/ml) was obtained in the plasma according to a plateau between 0.5 and 2 h. ( g – j ) TRP601 reduces excitotoxic lesions in neonates. The 5-day-old mice were subjected to intracerebral ibotenate injection and killed at different time points ( g =5 days; h and i =24 h; j =8 h) following the excitotoxic challenge to determine the impact of TRP601, TRP801, and TRP901 (1 mg/kg; i.p.) on lesion severity ( g ), microgliosis ( h ), astrogliosis ( i ), and group II caspases activity ( j ). Histograms show mean lesion volume ( g ; ▪, vehicle, n =16; TRP601, n =16; TRP801, n =7; TRP901, n =8), cell density ( h and i ; n =10 per group), or VDVADase activity ( j ; n =5 per group)±S.E.M. Asterisks indicate differences from control (▪) ( * P
    Active Recombinant Caspase 3, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    OriGene pure recombinant caspase 3
    KEA1–97 targets thioredoxin. (A) IsoTOP-ABPP analysis of KEA1–97 (10  μ M) in 231MFP breast cancer proteomes using DCT-alkyne probe. 231MFP breast cancer proteomes were pretreated with acetonitrile vehicle or KEA1–97 prior to labeling with DCT-alkyne (100  μ . (B) Gel-based ABPP studies showing competition of KEA1–97 against DCT-alkyne labeling (10  μ M) of pure human TXN. Shown is a representative gel and a dose−response curve. (C) TXN expression in GFP control or TXN-overexpressing 231MFP cells as assessed by qPCR. (D) Cell survival of 231MFP control or TXN-overexpressing cells treated with acetonitrile vehicle or KEA1–97 (10  μ M) for 24 h as assessed by Hoechst stain. (E) TXN activity assay. (F) Disruption of TXN interaction with caspase 3 upon treatment with KEA1–97 or mutation of TXN K72 to alanine. In the upper blot and left bar graph, pure His-tagged TXN and caspase 3 were preincubated with acetonitrile or KEA1–97 (100  μ M) prior to anti-His pulldown, SDS/PAGE, and blotting for caspase 3. In the lower blot and right bar graph, pure His-tagged TXN or TXN K72A mutant protein and caspase 3 were preincubated, subjected to anti-His pulldown, SDS/PAGE, and blotting for caspase 3. Data in B−E are shown as average ± SEM; data in A−F are  n  = 3/group. Significance is expressed as * p
    Pure Recombinant Caspase 3, supplied by OriGene, used in various techniques. Bioz Stars score: 83/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Becton Dickinson human recombinant caspase 3
    Transactivation response DNA-binding protein 43 (TDP-43) fragments distinguish cell death pathways. ( A ) Primary cortical neurons were pretreated with calpain (30 μ mol/L SNJ1945, SJ) and <t>caspase-3</t> (20 μ mol/L Z-VAD, ZD) inhibitors
    Human Recombinant Caspase 3, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 83/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Cloud-Clone human recombinant caspase 3
    Intracellular protein delivery. Intracellular delivery of <t>caspase</t> 3 in MCF-7 cells after 48 h incubation at a fixed PTX concentration of 10 μg/mL and caspase-3 concentration of 76 nM. (A) WB assay: (1) saline, (2) CLG, (3) naked caspase 3, (4) Taxol, (5) PNPs, (6) PNPplex, (7) HA-PNPplex, (8) PUL, (9) PULplex. (B) Quantitative analysis ( n = 3, * P
    Human Recombinant Caspase 3, supplied by Cloud-Clone, used in various techniques. Bioz Stars score: 84/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Addgene inc caspase assays recombinant caspase 3
    Relative activity of <t>caspase-8</t> with model peptides containing serine or phosphoserine at each position from P4 to P3′. Peptides modeled after the caspase-8 cleavage site of <t>pro-caspase-3</t> were incubated with caspase-8 (100 n m ) for 5 min at 37 °C before the reaction was terminated by an excess of the irreversible caspase inhibitor zVAD-fmk. Error bars represent the standard deviation of four reactions. Means of the caspase activities toward the phosphorylated and the corresponding nonphosphorylated peptide were compared using analysis of variance and Tukey's test. All pairs had p values
    Caspase Assays Recombinant Caspase 3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 83/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Santa Cruz Biotechnology recombinant caspase 3
    Relative activity of <t>caspase-8</t> with model peptides containing serine or phosphoserine at each position from P4 to P3′. Peptides modeled after the caspase-8 cleavage site of <t>pro-caspase-3</t> were incubated with caspase-8 (100 n m ) for 5 min at 37 °C before the reaction was terminated by an excess of the irreversible caspase inhibitor zVAD-fmk. Error bars represent the standard deviation of four reactions. Means of the caspase activities toward the phosphorylated and the corresponding nonphosphorylated peptide were compared using analysis of variance and Tukey's test. All pairs had p values
    Recombinant Caspase 3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 80/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Axxora active human recombinant caspase 3
    Cisplatin-induced p70S6K downregulation is mediated by <t>caspase-3.</t> (a), A549 cells were treated with 10 µM Mg132, 20 µM calpeptin and 20 µM z-VAD for 30 min prior to incubation with 30 μM cisplatin. (b), A549 cells were treated with 20 μM caspase inhibitors for 30 min prior to incubation with 30 μM cisplatin. z-VAD, broad specificity caspase inhibitor; z-VDVAD, caspase-2 inhibitor; z-DEVD, caspase-3 inhibitor; z-IETD, caspase-8 inhibitor; z-LEHD, caspase-9 inhibitor. Cells were processed following treatment with cisplatin for 24 h. Western blot analysis was performed with total cellular extracts using indicated antibodies. GAPDH was used as a loading control. The arrow indicates cleaved p70S6K band. Results are representative of two independent experiments. (c), A549 cells were transfected with control non-targeting siRNA or siRNA SMARTpool against caspase-2 or caspase-3 as described in “Experimental Procedures.” Western blot analysis was performed with total cellular extracts using indicated antibodies. (d), A549 cells transfected with control and caspase-2 siRNA were treated with or without cisplatin for 24 h and Western blot analysis was performed with total cell extracts. GAPDH was used as a loading control. The arrow indicates cleaved p70S6K band. Results are indicative of two independent experiments.
    Active Human Recombinant Caspase 3, supplied by Axxora, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Becton Dickinson human active recombinant caspase 3
    TDP-43 D169G mutant is more susceptible to proteolytic cleavage by caspase 3. ( A ) The wild-type, A90V and D169G mutant of TDP-43 (N-RRM12, residues 1–265) were incubated with <t>caspase</t> 3 for two hours. The SDS-PAGE stained with Coomassie blue revealed that TDP-43 was cleaved into TDP-35. Tandem mass spectrometry confirmed that caspase 3 digested N-RRM12 into two major fragments, TDP-35 (residues 90–265), and residues 1–89. ( B ) The wild-type, A90V and D169G mutant of TDP-43 (N-RRM12) were incubated with caspase 4, 6 and 7 for 16 hours. The digested proteins were resolved by 15% Tricine-SDS-PAGE with Coomassie blue staining. The mean percentages with standard errors shown at the bottom of the gel were calculated from three independent experiments. Statistical significance P values were determined by an unpaired two-tailed Student’s t -test.
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    BioVision recombinant caspase 3 positive control
    Protein expression of cleaved <t>caspase-3.</t> Western blots for cleaved caspase-3 in naïve, sham, hypoxia–ischemia injured+hypothermia (HI+HypoT), HI+rapid rewarming (HI+rapid RW), and HI+slow rewarming
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    Pharmingen active recombinant caspase 3
    Purified recombinant CK2 phosphorylates murine caspase-9 at Ser 348 in vitro . A , amino acid sequence in murine caspase-9 between the active site, QACGG ( * ), and the <t>caspase-3</t> processing site. Arrows show the auto-processing and primary caspase-8 cleavage site, SEPD, and the caspase-3 site, DQLD. The filled black circle shows serine 348 predicted to be the target residue within a strong CK2 consensus motif ( underlined ). B–D, in vitro translated mC-9 protein was immunoprecipitated with anti-caspase-9 antibody and incubated in kinase buffer in the presence or absence of purified CK2. B , phosphorylation of mC-9 in the presence of CK2 inhibitors ( lanes 3–5 ): DRB, heparin, and apigenin. C, top panel , CK2 phosphorylation of mC-9 and the LDAD mutant in the presence or absence of DRB in vitro. Lower panel , immunoprecipitation ( IP )/Western of cold in vitro translated mC-9 and LDAD. D , CK2 phosphorylation of mC-9 and mutants AEPD and LDVD in vitro .
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    BioVision active human recombinant caspase 3
    (A) Immunoblot for endogenous <t>pro-caspase-3</t> in hypoxic retinal cells treated without ( lane 2 ) and with enzyme inhibitors ( lanes 3–6 ). Intact pro-caspase-3 is indicated at 33 kDa ( filled arrowhead ) along with calpain-dependent breakdown products
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    Millipore caspase 3
    Fig. 3. GrB induces DNA fragmentation in the absence of <t>caspase-3</t> activation. ( A ) Structures of the caspase-3/-7-specific inhibitor (compound 1) and a 600-fold less active analog (compound 2). ( B ) Compound 1 blocks Fas-mediated apoptosis in a dose-dependent fashion. Jurkat cells were pre-treated with different concentrations of compound 1 (lanes 7–12) or compound 2 (lanes 1–6). Apoptosis was induced by cross-linking cell surface Fas receptors using mAb to Fas (M3, 20 µg/ml). Abrogation of Fas-induced apoptosis by compound 1 was monitored by DNA fragmentation, annexin V externalization, caspase-3 cleavage and DFF processing. No effect was noticed for compound 2 even at the highest concentration used (lanes 1–6). ( C ) DFF cleavage and DNA fragmentation occur in the absence of caspase-3 activation. Jurkat cells (1 × 10 6 cells per well) were pre-treated with 75 µM compound 1 or compound 2. Apoptosis by GrB/Ad 2 was induced as described in Materials and methods. Cell death was assayed as in (B). Pre-treatment of the cells with the caspase-3-specific inhibitor compound 1 did not prevent DFF processing and DNA fragmentation induced by GrB (lane 6). Pre-treatment of cells with the caspase-3-specific inhibitor compound 1 (lane 6), but not its analog compound 2 (lane 5), or DMSO alone (lane 4) resulted in a significant decrease in annexin V externalization. Untreated Jurkat cells (lane 1) and Jurkat cells treated with GrB or Ad 2 alone (lanes 2 and 3, respectively) showed no significant sign of apoptosis. These results are representative of two separate experiments. ( D ) Jurkat cells (1 × 10 5 cells per well) were pre-treated with 75 µM compound 1 or compound 2, and apoptosis was induced by recombinant GrB/perforin as described in Materials and methods. Cell death was assayed by 51 Cr release. Pre-treatment with compound 1, but not compound 2, or DMSO blocked further processing of caspase-3 to the p17 active form (lanes 6, 4 and 5, respectively, upper panel) and processing of caspase-mediated cleavage of Rho-GDI substrate (lanes 6, 4 and 5, respectively, lower panel). Blockage of caspase-3 activity had no effect on DFF processing (lane 6, middle panel). Untreated Jurkat cells (lane 1) and those treated with perforin or GrB alone (lanes 2 and 3, respectively) showed no significant sign of apoptosis. These results are representative of three separate experiments.
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    Axxora rec caspase 3
    Purified and lysate-associated c-gelsolin is cleaved by rec.gzmB independent of caspase 3. A  and  B , rec.c-gelsolin was treated with rec.gzmB in a dose ( A )- and time-dependent manner ( B ).  C , specific proteolysis of c-gelsolin by gzmB was verified using
    Rec Caspase 3, supplied by Axxora, used in various techniques. Bioz Stars score: 80/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pharmingen human recombinant caspase 3
    Immunofluorescent dopaminergic neurons in primary cultures of rat mesencephalic neurons fixed after 12 hr of 1-μM MPP + treatment stained with a TH monoclonal mouse antibody revealed by FITC ( A ) and a rabbit polyclonal antibody recognizing activated <t>caspase-3</t> (CM1) revealed by tetramethylrhodamine B isothiocyanate ( B ). Hoechst 33258 staining reveals an intact nucleus in this neuron ( C , arrow). (Bar = 20 μm.) In contrast, in a TH-positive neuron ( D ) with CM1 staining ( E ), DNA condensation assessed by Hoechst 33258 can be detected after 72 hr of MPP + treatment ( F , arrow). Note that a higher magnification was used to show DNA condensation. (Bar = 10 μm.)
    Human Recombinant Caspase 3, supplied by Pharmingen, used in various techniques. Bioz Stars score: 78/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA human recombinant caspase 3
    Abolition of <t>caspase-3</t> cleavage of PARP by mutation at the cleavage site (DEVD). (A) The full-length human PARP (hPARP) cDNA was under control of the T7 and SV40 promoters (PARP/WT). An Asp (D)→Asn (N) transition (codon 214, asterisk) was introduced into the DEVD site, resulting in a mutant construct designated D214N. The polyadenylation signal [(poly (A)] was from SV40. MCS, multiple cloning sites. The arrow indicates the caspase cleavage site. (B) [ 35 S]methionine-labeled PARP was generated by an in vitro coupled transcription-translation system. Translated products from PARP/WT and D214N vectors were incubated either with (+) or without (−) purified human recombinant caspase-3. (C) Western blot analysis of PARP expression and cleavage in transfected fibroblasts. Stably transfected clones expressing wild-type PARP (WT/2 and WT/3) and those expressing mutant PARP (DN/8 and DN/10) were either treated or not treated with TNF-α (20 ng/ml); this was followed by Western blot analysis with polyclonal antiserum Vic-5. Full-length PARP (113 kDa) and two cleaved fragments (89 and 24 kDa) are indicated by arrowheads.
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    Enzo Biochem human recombinant caspase 3
    Abolition of <t>caspase-3</t> cleavage of PARP by mutation at the cleavage site (DEVD). (A) The full-length human PARP (hPARP) cDNA was under control of the T7 and SV40 promoters (PARP/WT). An Asp (D)→Asn (N) transition (codon 214, asterisk) was introduced into the DEVD site, resulting in a mutant construct designated D214N. The polyadenylation signal [(poly (A)] was from SV40. MCS, multiple cloning sites. The arrow indicates the caspase cleavage site. (B) [ 35 S]methionine-labeled PARP was generated by an in vitro coupled transcription-translation system. Translated products from PARP/WT and D214N vectors were incubated either with (+) or without (−) purified human recombinant caspase-3. (C) Western blot analysis of PARP expression and cleavage in transfected fibroblasts. Stably transfected clones expressing wild-type PARP (WT/2 and WT/3) and those expressing mutant PARP (DN/8 and DN/10) were either treated or not treated with TNF-α (20 ng/ml); this was followed by Western blot analysis with polyclonal antiserum Vic-5. Full-length PARP (113 kDa) and two cleaved fragments (89 and 24 kDa) are indicated by arrowheads.
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    R&D Systems human recombinant caspase 3
    In vitro proof of rASC apoptosis at 24 h after incubation with Mitomycin (MMC) (a) Confocal microscopy of viable and MMC treated, apoptotic rASCs demonstrates cleaved <t>Caspase-3</t> (green fluorescent indicates activated caspase-3; red fluorescent shows F-actin filaments; and DAPI displays cell nucleus with blue fluorescent). (b) Flow cytometry (Ex/Em 488/530) analysis of viable (left panel) and apoptotic rASCs (right panel) which were stained with FLICA kit (fluorescent inhibitor of caspases). (c) Quantification of cleaved caspase-3 in viable and apoptotic rASCs by the SensoLyte Homogenous AMC caspase-3/7 kit. Upon caspase-3/7 cleavage, Ac-DEVD-AMC generates the AMC fluorophore which has bright blue fluorescence and can be quantified at Ex/Em=354 nm/442 nm. Data are displayed as means of three experiments in each group with standard errors. *** indicates significant differences between viable and apoptotic cells ( P
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    Image Search Results


    ( A ) Ligation of (1–45)αCOSR and (46–142) at 1.5 h. The reaction was monitored by analytical HPLC on a Waters XBridge C 18 column (4.6 × 150 mm, 3.5 μM) running a 30-min gradient of 25%–45% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. ( Insets ) Mass spectra of (1–45)αCOSR and (46–142) determined by ESI-MS. ( B ) Ligated full-length survivin characterized by C 18 RP-HPLC and ESI-MS. HPLC conditions: Waters symmetry 300 C 18 column (4.6 × 150 mm, 5 μM) running a 30-min gradient of 5%–65% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. ( C ) CD spectra of synthetic survivin at 5 μM in 5 mM phosphate buffer containing 0.1 mM TCEP, pH 7.5 (thin line), and at 25 μM in 5 mM phosphate buffer containing 0.1 mM TCEP and 50 μM Zn 2+ , pH 7.5 (thick line). ( D ) Representative size-exclusion chromatograms of synthetic survivin (3) and molecular mass standards (1, 2, 4, 5). Linear regression analysis of the correlation between logarithmic M r and retention time is illustrated in the inset . ( E ) Representative raw data from the hydrolysis of Ac-DEVD-AMC by caspase-3 in the absence and presence of different concentrations of XIAP and synthetic survivin. ( F ) Dose-dependent percent inhibition of caspase-3 by XIAP (filled circles), synthetic survivin without Zn 2+ (empty squares), and synthetic survivin in the presence of Zn 2+ (filled squares). Each curve is the mean of three independent experiments.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Chemically synthesized human survivin does not inhibit caspase-3

    doi: 10.1110/ps.036145.108

    Figure Lengend Snippet: ( A ) Ligation of (1–45)αCOSR and (46–142) at 1.5 h. The reaction was monitored by analytical HPLC on a Waters XBridge C 18 column (4.6 × 150 mm, 3.5 μM) running a 30-min gradient of 25%–45% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. ( Insets ) Mass spectra of (1–45)αCOSR and (46–142) determined by ESI-MS. ( B ) Ligated full-length survivin characterized by C 18 RP-HPLC and ESI-MS. HPLC conditions: Waters symmetry 300 C 18 column (4.6 × 150 mm, 5 μM) running a 30-min gradient of 5%–65% acetonitrile containing 0.1% TFA at a flow rate of 1 mL/min. ( C ) CD spectra of synthetic survivin at 5 μM in 5 mM phosphate buffer containing 0.1 mM TCEP, pH 7.5 (thin line), and at 25 μM in 5 mM phosphate buffer containing 0.1 mM TCEP and 50 μM Zn 2+ , pH 7.5 (thick line). ( D ) Representative size-exclusion chromatograms of synthetic survivin (3) and molecular mass standards (1, 2, 4, 5). Linear regression analysis of the correlation between logarithmic M r and retention time is illustrated in the inset . ( E ) Representative raw data from the hydrolysis of Ac-DEVD-AMC by caspase-3 in the absence and presence of different concentrations of XIAP and synthetic survivin. ( F ) Dose-dependent percent inhibition of caspase-3 by XIAP (filled circles), synthetic survivin without Zn 2+ (empty squares), and synthetic survivin in the presence of Zn 2+ (filled squares). Each curve is the mean of three independent experiments.

    Article Snippet: EnzChek Caspase-3 assay kit #1 was purchased from Invitrogen; recombinant caspase-3 was obtained from Calbiochem and recombinant human XIAP, from R & D Systems.

    Techniques: Ligation, High Performance Liquid Chromatography, Flow Cytometry, Mass Spectrometry, Inhibition

    Relative resistance of PrP C from mouse brain lysate to calpain proteolysis . Lysate from naïve mouse brains were prepared and digested with calpain-1, -2, or caspase-3 as described in section “Materials and Methods.” Aliquots of the untreated and treated lysates were then electrophoresed, western blotted, and immunostained with either anti-αII-spectrin Ab (A) or with anti-PrP Mab E11. (B) Characteristic calpain-generated SBDP150 and SBDP145 as well as caspase-3 generated SBDP150i and SBDP120 are indicated. No PrP C -BDPs were observed following the treatments with Mab 7E4 (data not shown).

    Journal: Frontiers in Neurology

    Article Title: Release of Full-Length PrPC from Cultured Neurons Following Neurotoxic Challenge

    doi: 10.3389/fneur.2012.00147

    Figure Lengend Snippet: Relative resistance of PrP C from mouse brain lysate to calpain proteolysis . Lysate from naïve mouse brains were prepared and digested with calpain-1, -2, or caspase-3 as described in section “Materials and Methods.” Aliquots of the untreated and treated lysates were then electrophoresed, western blotted, and immunostained with either anti-αII-spectrin Ab (A) or with anti-PrP Mab E11. (B) Characteristic calpain-generated SBDP150 and SBDP145 as well as caspase-3 generated SBDP150i and SBDP120 are indicated. No PrP C -BDPs were observed following the treatments with Mab 7E4 (data not shown).

    Article Snippet: Human recombinant calpain-1, rat calpain-2, and human recombinant caspase-3 were from EMD Millipore Biosciences (Billerica, MA, USA).

    Techniques: Western Blot, Generated

    Release of full-length PrP C into culture media from RtCNC challenged with neurotoxins MTX and NMDA . RtCNC were either untreated (control; Ctrl) or challenged with MTX or NMDA. (A) Soluble and insoluble cell fractions and conditioned cell culture media were analyzed by SDS-PAGE followed by western blotting and immunostaining with Mab D8. (B) Soluble cell fractions were also analyzed with anti-αll spectrin Mab to probe the formation of SBDPs (SBDP150 by calpain and SBDP120 by caspase-3). Blots are representative of four separate experiments.

    Journal: Frontiers in Neurology

    Article Title: Release of Full-Length PrPC from Cultured Neurons Following Neurotoxic Challenge

    doi: 10.3389/fneur.2012.00147

    Figure Lengend Snippet: Release of full-length PrP C into culture media from RtCNC challenged with neurotoxins MTX and NMDA . RtCNC were either untreated (control; Ctrl) or challenged with MTX or NMDA. (A) Soluble and insoluble cell fractions and conditioned cell culture media were analyzed by SDS-PAGE followed by western blotting and immunostaining with Mab D8. (B) Soluble cell fractions were also analyzed with anti-αll spectrin Mab to probe the formation of SBDPs (SBDP150 by calpain and SBDP120 by caspase-3). Blots are representative of four separate experiments.

    Article Snippet: Human recombinant calpain-1, rat calpain-2, and human recombinant caspase-3 were from EMD Millipore Biosciences (Billerica, MA, USA).

    Techniques: Cell Culture, SDS Page, Western Blot, Immunostaining

    HrPrP vulnerability to calpain-1, -2 digestion with identification of major cleavage sites . E. coli expressed recombinant N-terminal (His) 6 -tag fusion protein with human PrP C residues 23–231 (Human prion protein; accession # NP 898902) was subjected to calpain-1, calpain-2, and caspase-3 digestion (1:50 protease:substrate ratio). Following SDS-PAGE, proteins were transferred to PVDF membrane and stained with Coomassie blue. (A) Major BDPs were identified by N-terminal microsequencing. (B) Based on the PrP C amino acid sequence the cleavage sites are identified and depicted by downward arrows. The new N-terminal sequences are identified in bold and the calpain recognition P1–P2 sequences are underlined and in bold (C) .

    Journal: Frontiers in Neurology

    Article Title: Release of Full-Length PrPC from Cultured Neurons Following Neurotoxic Challenge

    doi: 10.3389/fneur.2012.00147

    Figure Lengend Snippet: HrPrP vulnerability to calpain-1, -2 digestion with identification of major cleavage sites . E. coli expressed recombinant N-terminal (His) 6 -tag fusion protein with human PrP C residues 23–231 (Human prion protein; accession # NP 898902) was subjected to calpain-1, calpain-2, and caspase-3 digestion (1:50 protease:substrate ratio). Following SDS-PAGE, proteins were transferred to PVDF membrane and stained with Coomassie blue. (A) Major BDPs were identified by N-terminal microsequencing. (B) Based on the PrP C amino acid sequence the cleavage sites are identified and depicted by downward arrows. The new N-terminal sequences are identified in bold and the calpain recognition P1–P2 sequences are underlined and in bold (C) .

    Article Snippet: Human recombinant calpain-1, rat calpain-2, and human recombinant caspase-3 were from EMD Millipore Biosciences (Billerica, MA, USA).

    Techniques: Recombinant, SDS Page, Staining, Sequencing

    Examination of caspase activity in cocultures incubated under simulated ischemia or elevated hydrostatic pressure. A , Western blot analysis of caspase-8 expression in cocultures. B , Western blot analysis of caspase-3 expression cocultures. Column 1 , Control retinal ganglion cells; column 2 , retinal ganglion cells incubated under simulated ischemia for 72 hr; column 3 , retinal ganglion cells incubated under elevated hydrostatic pressure for 72 hr; column 4 , control glial cells; column 5 , glial cells incubated under simulated ischemia for 72 hr; column 6 , glial cells incubated under elevated hydrostatic pressure for 72 hr. Western blots revealed that the 55 kDa immunoreactive band corresponding to caspase-8 cleaved to 30 and 20 kDa products in retinal ganglion cells that were incubated under stress conditions. In addition, 32 kDa pro-enzyme caspase-3 cleaved to a 17 kDa active subunit in retinal ganglion cells. No cleavage of caspase-8 or caspase-3 was detected with the use of the extracts of glial cells. Caspase activation also was examined, in situ , using Phiphilux-G 6 D 2 in retinal ganglion cells that were incubated under different conditions for 72 hr. C , Normal conditions. D , Simulated ischemia. E , Elevated hydrostatic pressure. Fluorescence microscope images seen in F–H correspond to phase-contrast images of the retinal ganglion cells seen in C–E , respectively. Rhodamine fluorescence ( red ) indicates caspase-3-like activity in retinal ganglion cells that were incubated under stress conditions. I , The amount of DEVD-AMC cleaving activity with the use of fluorometric analysis was higher in retinal ganglion cells in cocultures that were incubated under simulated ischemia or elevated hydrostatic pressure for 72 hr as compared with cocultures that were incubated under normal conditions (Mann–Whitney U test; p = 0.006 and p = 0.04, respectively).

    Journal: The Journal of Neuroscience

    Article Title: Increased Production of Tumor Necrosis Factor-α by Glial Cells Exposed to Simulated Ischemia or Elevated Hydrostatic Pressure Induces Apoptosis in Cocultured Retinal Ganglion Cells

    doi: 10.1523/JNEUROSCI.20-23-08693.2000

    Figure Lengend Snippet: Examination of caspase activity in cocultures incubated under simulated ischemia or elevated hydrostatic pressure. A , Western blot analysis of caspase-8 expression in cocultures. B , Western blot analysis of caspase-3 expression cocultures. Column 1 , Control retinal ganglion cells; column 2 , retinal ganglion cells incubated under simulated ischemia for 72 hr; column 3 , retinal ganglion cells incubated under elevated hydrostatic pressure for 72 hr; column 4 , control glial cells; column 5 , glial cells incubated under simulated ischemia for 72 hr; column 6 , glial cells incubated under elevated hydrostatic pressure for 72 hr. Western blots revealed that the 55 kDa immunoreactive band corresponding to caspase-8 cleaved to 30 and 20 kDa products in retinal ganglion cells that were incubated under stress conditions. In addition, 32 kDa pro-enzyme caspase-3 cleaved to a 17 kDa active subunit in retinal ganglion cells. No cleavage of caspase-8 or caspase-3 was detected with the use of the extracts of glial cells. Caspase activation also was examined, in situ , using Phiphilux-G 6 D 2 in retinal ganglion cells that were incubated under different conditions for 72 hr. C , Normal conditions. D , Simulated ischemia. E , Elevated hydrostatic pressure. Fluorescence microscope images seen in F–H correspond to phase-contrast images of the retinal ganglion cells seen in C–E , respectively. Rhodamine fluorescence ( red ) indicates caspase-3-like activity in retinal ganglion cells that were incubated under stress conditions. I , The amount of DEVD-AMC cleaving activity with the use of fluorometric analysis was higher in retinal ganglion cells in cocultures that were incubated under simulated ischemia or elevated hydrostatic pressure for 72 hr as compared with cocultures that were incubated under normal conditions (Mann–Whitney U test; p = 0.006 and p = 0.04, respectively).

    Article Snippet: Positive controls included purified recombinant caspase-3 (0.1 μg; Upstate Biotechnology, Lake Placid, NY).

    Techniques: Activity Assay, Incubation, Western Blot, Expressing, Activation Assay, In Situ, Fluorescence, Microscopy, MANN-WHITNEY

    GRASP65 cleavage is an early event during apoptosis. ( a ) Time-lapse imaging of GRASP65–GFP caspase cleavage. To the top, phase contrast images of a TRAIL-treated HeLa cell before and after the onset of apoptosis; to the bottom, fluorescence images of Golgi-associated GRASP65–GFP and the loss of GFP signal from the Golgi following the onset of apoptosis. ( b ) Normalised fluorescence intensity traces of Golgi-associated GRASP65–GFP in HeLa cells treated with the apoptosis-inducing reagents shown. Traces are plotted relative to the onset of cell rounding (hatched boxes). Data show means ± S.D. for a minimum of five cells per treatment. ( c ) Autoradiographs of 35 S-labelled GRASP65 translated in vitro and incubated for 90 min with increasing concentrations of recombinant caspase-3 or caspase-8. Full-length (

    Journal: Cell Death & Disease

    Article Title: Caspase cleavage of the Golgi stacking factor GRASP65 is required for Fas/CD95-mediated apoptosis

    doi: 10.1038/cddis.2010.59

    Figure Lengend Snippet: GRASP65 cleavage is an early event during apoptosis. ( a ) Time-lapse imaging of GRASP65–GFP caspase cleavage. To the top, phase contrast images of a TRAIL-treated HeLa cell before and after the onset of apoptosis; to the bottom, fluorescence images of Golgi-associated GRASP65–GFP and the loss of GFP signal from the Golgi following the onset of apoptosis. ( b ) Normalised fluorescence intensity traces of Golgi-associated GRASP65–GFP in HeLa cells treated with the apoptosis-inducing reagents shown. Traces are plotted relative to the onset of cell rounding (hatched boxes). Data show means ± S.D. for a minimum of five cells per treatment. ( c ) Autoradiographs of 35 S-labelled GRASP65 translated in vitro and incubated for 90 min with increasing concentrations of recombinant caspase-3 or caspase-8. Full-length (

    Article Snippet: In vitro caspase cleavage of GRASP65 was carried out using 35 S-labelled, in vitro translated GRASP65 and recombinant caspase-3 and caspase-8 (Alexis Biochemicals) as described in Lane et al. Products were incubated in caspase buffer (0.1% CHAPS, 10% sucrose, 5 mM DTT, 2 mM EDTA, 50 mM Hepes, pH 7.4) for 90 min at 30°C and run on SDS-PAGE gels for autoradiography.

    Techniques: Imaging, Fluorescence, In Vitro, Incubation, Recombinant

    Kaempferol maintains cIAPs and inhibits caspase 3/7 activation. ( a , b ) Expression of cIAP1 and cIAP2 mRNA level was measured by q-RTPCR. ER stress was induced in IMR32 cells with BFA in the presence and absence of kaempferol pre-treatment. Expression of cIAPs level was compared to caspase 3/7 activation in 12 h and 24 h of ER stress induction. Data represented as average ± SEM of two independent experiments performed in duplicate.  * Represents the comparison of BFA treated condition to control cells;  # represents the significant difference between BFA treated cells to BFA + kaempferol treated cells at p ≤ 0.05. ( c ) Regulation cIAPs mRNA level at increasing treatment hours in IMR32 cells cultured with BFA with/without kaempferol pre-treatment. Data represented as average ± SEM of two independent experiments performed in duplicate. ( d,e ) Protein levels of cIAP1 and cIAP2 in IMR32 cells after 24 hours of treatment with BFA and kaempferol pre-treated conditions by immunocytochemistry (Scale bar: 67 µm). ( f ) MTT assay showing the reversal of cell death protection by SMAC mimetic compounds co-treatment with KFL + BFA treated cells after 24 hours of incubation. ( g ) SMAC mimetic increases the caspase 3/7 activity in IMR32 cells upon treated with BFA alone and in BFA + KFL combination. Data represented as average ±SEM of two independent experiments performed in triplicate. * and  $  Represents significance at p 

    Journal: Scientific Reports

    Article Title: Kaempferol mitigates Endoplasmic Reticulum Stress Induced Cell Death by targeting caspase 3/7

    doi: 10.1038/s41598-018-20499-7

    Figure Lengend Snippet: Kaempferol maintains cIAPs and inhibits caspase 3/7 activation. ( a , b ) Expression of cIAP1 and cIAP2 mRNA level was measured by q-RTPCR. ER stress was induced in IMR32 cells with BFA in the presence and absence of kaempferol pre-treatment. Expression of cIAPs level was compared to caspase 3/7 activation in 12 h and 24 h of ER stress induction. Data represented as average ± SEM of two independent experiments performed in duplicate. * Represents the comparison of BFA treated condition to control cells; # represents the significant difference between BFA treated cells to BFA + kaempferol treated cells at p ≤ 0.05. ( c ) Regulation cIAPs mRNA level at increasing treatment hours in IMR32 cells cultured with BFA with/without kaempferol pre-treatment. Data represented as average ± SEM of two independent experiments performed in duplicate. ( d,e ) Protein levels of cIAP1 and cIAP2 in IMR32 cells after 24 hours of treatment with BFA and kaempferol pre-treated conditions by immunocytochemistry (Scale bar: 67 µm). ( f ) MTT assay showing the reversal of cell death protection by SMAC mimetic compounds co-treatment with KFL + BFA treated cells after 24 hours of incubation. ( g ) SMAC mimetic increases the caspase 3/7 activity in IMR32 cells upon treated with BFA alone and in BFA + KFL combination. Data represented as average ±SEM of two independent experiments performed in triplicate. * and $ Represents significance at p 

    Article Snippet: For enzyme inhibition assays, recombinant Caspase 3 enzyme was obtained from Enzo Life sciences, BIOMOL Cat. #SE-169 and Caspase 3/7 glo assay reagent from Promega.

    Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Immunocytochemistry, MTT Assay, Incubation, Activity Assay

    Graphical abstract. Kaempferol alleviates ER stress by inhibiting the executioner caspase 3/7. Endogenous inhibitor of apoptotic proteins-IAPs level did not get altered upon kaempferol treatment and the SMAC mimetics which are inhibitor of IAPs reverses the kaempferol mediated caspase inhibition and allows cell death to occur.

    Journal: Scientific Reports

    Article Title: Kaempferol mitigates Endoplasmic Reticulum Stress Induced Cell Death by targeting caspase 3/7

    doi: 10.1038/s41598-018-20499-7

    Figure Lengend Snippet: Graphical abstract. Kaempferol alleviates ER stress by inhibiting the executioner caspase 3/7. Endogenous inhibitor of apoptotic proteins-IAPs level did not get altered upon kaempferol treatment and the SMAC mimetics which are inhibitor of IAPs reverses the kaempferol mediated caspase inhibition and allows cell death to occur.

    Article Snippet: For enzyme inhibition assays, recombinant Caspase 3 enzyme was obtained from Enzo Life sciences, BIOMOL Cat. #SE-169 and Caspase 3/7 glo assay reagent from Promega.

    Techniques: Inhibition

    Kaempferol rescues cells against BFA induced cell death. ( a ) Screening for cytoprotective compounds (50 µM) against BFA (1 µg/ml) induced ER stress in IMR32 cell line. Cells were plated and pre-incubated with the test compounds (90 min) and treated with ER stress inducer BFA, incubated for 24 h. Cell viability was estimated by MTT assay. BFA: Brefeldin A; Withan: Withanone; With A: Withanaloide A; With B: Withanaloide B; kaemp: kaempferol; kaempG: Kaempfeol-3-O-robinbioside-7-O-glucoside; Bacop I: Bacopaside I; PEA: Palmitoylethanolamide; PBA: Phenyl Butyric acid; Sal: Salubrinal. ( b ) Confirmation of cytoprotective activity of kaempferol by measuring cellular ATP level using CellTitre Glo reagent. ( c ) Phase contrast images of IMR32 cells after BFA treatment in presence or absence of kaempferol pre-incubation. ( d–f ) Cytoprotective activity of kaempferol against BFA induced cell death in multiple cell lines with incubation time of 24 hours. Cell viability was assessed using MTT assay. ( g ) Inhibition of caspase 3/7 activation by kaempferol against BFA and CDDO-Me induced caspase 3/7 activation in IMR32 cells. Data represents mean ± SEM of experiment performed in triplicate (n = 3). Note: *Represents the significance between cell death inducer alone treated condition compared to kaempferol pre-treated condition, at p ≤ 0.05 (one way ANOVA).

    Journal: Scientific Reports

    Article Title: Kaempferol mitigates Endoplasmic Reticulum Stress Induced Cell Death by targeting caspase 3/7

    doi: 10.1038/s41598-018-20499-7

    Figure Lengend Snippet: Kaempferol rescues cells against BFA induced cell death. ( a ) Screening for cytoprotective compounds (50 µM) against BFA (1 µg/ml) induced ER stress in IMR32 cell line. Cells were plated and pre-incubated with the test compounds (90 min) and treated with ER stress inducer BFA, incubated for 24 h. Cell viability was estimated by MTT assay. BFA: Brefeldin A; Withan: Withanone; With A: Withanaloide A; With B: Withanaloide B; kaemp: kaempferol; kaempG: Kaempfeol-3-O-robinbioside-7-O-glucoside; Bacop I: Bacopaside I; PEA: Palmitoylethanolamide; PBA: Phenyl Butyric acid; Sal: Salubrinal. ( b ) Confirmation of cytoprotective activity of kaempferol by measuring cellular ATP level using CellTitre Glo reagent. ( c ) Phase contrast images of IMR32 cells after BFA treatment in presence or absence of kaempferol pre-incubation. ( d–f ) Cytoprotective activity of kaempferol against BFA induced cell death in multiple cell lines with incubation time of 24 hours. Cell viability was assessed using MTT assay. ( g ) Inhibition of caspase 3/7 activation by kaempferol against BFA and CDDO-Me induced caspase 3/7 activation in IMR32 cells. Data represents mean ± SEM of experiment performed in triplicate (n = 3). Note: *Represents the significance between cell death inducer alone treated condition compared to kaempferol pre-treated condition, at p ≤ 0.05 (one way ANOVA).

    Article Snippet: For enzyme inhibition assays, recombinant Caspase 3 enzyme was obtained from Enzo Life sciences, BIOMOL Cat. #SE-169 and Caspase 3/7 glo assay reagent from Promega.

    Techniques: Incubation, MTT Assay, Activity Assay, Inhibition, Activation Assay

    Caspase cleavage of pBim EL in T cells at an early stage of apoptosis. ( A ) Caspase cleavage of pBim EL in apoptotic Jurkat T cells. Anti-Bim Western blotting was performed with the rabbit polyclonal antibodies directed against the full-length Bim EL to examine the purified Flag-tagged rBim EL and Flag-tagged rBim L (lanes 1 and 2), Jurkat cell lysates incubated with (lane 5) or without (lanes 3 and 4) PAP, and lysates from Jurkat cells subjected to the various treatments as indicated (lanes 7–20). In lanes 19 and 20, the antibody solution was preincubated with purified rBim EL before being used in Western blotting. In lanes 21–24, Western blotting was performed with anti-Bim antibodies obtained from StressGen Biotechnologies (Victoria, Canada) and ProMab Biotechnologies (Mountain View, CA), respectively. ( B ) Cleavage of pBim EL at an early stage of apoptosis. Lysates of Jurkat cells treated with staurosporine for the indicated periods of time (hr) were analyzed by anti-Bim Western blotting. The percentage of apoptotic cells was determined by flow-cytometric analysis and is indicated at the bottom. ( C ) Apoptosis-induced cleavage of pBim EL in activated mouse primary T cells. Anti-Bim Western blot analysis of lysates from activated mouse primary T cells treated with or without staurosporine. ( D ) pBim EL cleaved in vitro by recombinant caspases-3 comigrates with tBim EL from apoptotic Jurkat cells as indicated by anti-Bim Western blotting. Lanes 1 and 2, lysates from healthy Jurkat cells were incubated with or without recombinant caspase-3. Lanes 3 and 4 show lysates from Jurkat cells treated with or without staurosporine. Stau, staurosporine; OA, okadaic acid; mBim EL , modified Bim EL .

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

    Article Title: Caspase cleavage of BimEL triggers a positive feedback amplification of apoptotic signaling

    doi: 10.1073/pnas.0308050100

    Figure Lengend Snippet: Caspase cleavage of pBim EL in T cells at an early stage of apoptosis. ( A ) Caspase cleavage of pBim EL in apoptotic Jurkat T cells. Anti-Bim Western blotting was performed with the rabbit polyclonal antibodies directed against the full-length Bim EL to examine the purified Flag-tagged rBim EL and Flag-tagged rBim L (lanes 1 and 2), Jurkat cell lysates incubated with (lane 5) or without (lanes 3 and 4) PAP, and lysates from Jurkat cells subjected to the various treatments as indicated (lanes 7–20). In lanes 19 and 20, the antibody solution was preincubated with purified rBim EL before being used in Western blotting. In lanes 21–24, Western blotting was performed with anti-Bim antibodies obtained from StressGen Biotechnologies (Victoria, Canada) and ProMab Biotechnologies (Mountain View, CA), respectively. ( B ) Cleavage of pBim EL at an early stage of apoptosis. Lysates of Jurkat cells treated with staurosporine for the indicated periods of time (hr) were analyzed by anti-Bim Western blotting. The percentage of apoptotic cells was determined by flow-cytometric analysis and is indicated at the bottom. ( C ) Apoptosis-induced cleavage of pBim EL in activated mouse primary T cells. Anti-Bim Western blot analysis of lysates from activated mouse primary T cells treated with or without staurosporine. ( D ) pBim EL cleaved in vitro by recombinant caspases-3 comigrates with tBim EL from apoptotic Jurkat cells as indicated by anti-Bim Western blotting. Lanes 1 and 2, lysates from healthy Jurkat cells were incubated with or without recombinant caspase-3. Lanes 3 and 4 show lysates from Jurkat cells treated with or without staurosporine. Stau, staurosporine; OA, okadaic acid; mBim EL , modified Bim EL .

    Article Snippet: In vitro cleavage coupled with microtubule assembly was carried out by incubating the cleared Jurkat cell lysate (from 1 × 106 cells) with 5 μg/ml taxol at 37°C for 20 min. After microtubules were assembled, 100 units of recombinant caspase-3 (Calbiochem) were added.

    Techniques: Western Blot, Purification, Incubation, Flow Cytometry, In Vitro, Recombinant, Modification

    N-terminal cleavage of Bim EL after Asp-13 enhances its interaction with Bcl-2 and induction of apoptosis. ( A ) In vivo caspase-cleaved tBim EL comigrates with rBim EL ΔN13 in SDS gel. Bim EL truncated after the various Asp residues were expressed in transfected 293T cells. Their mobility was compared with that of tBim EL from staurosporine (Stau)-treated Jurkat cells by Western blotting. ( B ) Mutation of Asp-13 to Asn in Bim EL prevents cleavage by caspase-3. Anti-Bim Western blot analysis of recombinant wild-type (WT) and mutant (D7N and D13N) Bim EL before (–) or after (+) the incubation with recombinant caspase-3. ( C ) Expression of ΔN13 greatly reduces cell survival. Jurkat cells infected with retroviruses expressing full-length Flag-tagged Bim EL (FL Bim EL -Flag), Flag-tagged ΔN13 (ΔN13-Flag), or nothing (vector) were selected with puromycin at 2 days after infection. The percentage of surviving cells was determined over the subsequent 8 days, and the average from three independent infections is shown ( Left ). Flag-tagged Bim EL and Flag-tagged ΔN13 produced in Phoenix retrovirus packaging cells from three independent transfections were analyzed by anti-Bim Western blotting, which also detected the endogenous Bim EL ( Right ). ( D ) ΔN13 has greater apoptotic activity than wild-type Bim EL . Plasmids expressing Flag-tagged Bim EL or Flag-tagged ΔN13 were electroporated into Jurkat cells. Apoptosis was measured by flow-cytometric analysis of annexin V-positive and propidium iodide-negative cells. ( E ) ΔN13 binds more Bcl-2 than Flag-tagged Bim EL or control. Flag-tagged Bim EL , Flag-tagged ΔN13, and their associated Bcl-2 were immunoprecipitated (α-Flag IP) from the lysates of transfected Jurkat cells and analyzed by Western blotting as indicated.

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

    Article Title: Caspase cleavage of BimEL triggers a positive feedback amplification of apoptotic signaling

    doi: 10.1073/pnas.0308050100

    Figure Lengend Snippet: N-terminal cleavage of Bim EL after Asp-13 enhances its interaction with Bcl-2 and induction of apoptosis. ( A ) In vivo caspase-cleaved tBim EL comigrates with rBim EL ΔN13 in SDS gel. Bim EL truncated after the various Asp residues were expressed in transfected 293T cells. Their mobility was compared with that of tBim EL from staurosporine (Stau)-treated Jurkat cells by Western blotting. ( B ) Mutation of Asp-13 to Asn in Bim EL prevents cleavage by caspase-3. Anti-Bim Western blot analysis of recombinant wild-type (WT) and mutant (D7N and D13N) Bim EL before (–) or after (+) the incubation with recombinant caspase-3. ( C ) Expression of ΔN13 greatly reduces cell survival. Jurkat cells infected with retroviruses expressing full-length Flag-tagged Bim EL (FL Bim EL -Flag), Flag-tagged ΔN13 (ΔN13-Flag), or nothing (vector) were selected with puromycin at 2 days after infection. The percentage of surviving cells was determined over the subsequent 8 days, and the average from three independent infections is shown ( Left ). Flag-tagged Bim EL and Flag-tagged ΔN13 produced in Phoenix retrovirus packaging cells from three independent transfections were analyzed by anti-Bim Western blotting, which also detected the endogenous Bim EL ( Right ). ( D ) ΔN13 has greater apoptotic activity than wild-type Bim EL . Plasmids expressing Flag-tagged Bim EL or Flag-tagged ΔN13 were electroporated into Jurkat cells. Apoptosis was measured by flow-cytometric analysis of annexin V-positive and propidium iodide-negative cells. ( E ) ΔN13 binds more Bcl-2 than Flag-tagged Bim EL or control. Flag-tagged Bim EL , Flag-tagged ΔN13, and their associated Bcl-2 were immunoprecipitated (α-Flag IP) from the lysates of transfected Jurkat cells and analyzed by Western blotting as indicated.

    Article Snippet: In vitro cleavage coupled with microtubule assembly was carried out by incubating the cleared Jurkat cell lysate (from 1 × 106 cells) with 5 μg/ml taxol at 37°C for 20 min. After microtubules were assembled, 100 units of recombinant caspase-3 (Calbiochem) were added.

    Techniques: In Vivo, SDS-Gel, Transfection, Western Blot, Mutagenesis, Recombinant, Incubation, Expressing, Infection, Plasmid Preparation, Produced, Activity Assay, Flow Cytometry, Immunoprecipitation

    pBim EL escapes sequestration by microtubules and becomes accessible to caspase cleavage. ( A ) pBim EL is released from microtubules. Jurkat cell lysate preincubated with or without PAP ( Left ) or lysate from Jurkat cells treated with or without okadaic acid ( Right ) was incubated with taxol and then subjected to ultracentrifugation. α-Tubulin and Bim in the pellet (P), supernatant (S), or lysate before centrifugation (T) were detected by Western blotting. ( B ) The association with microtubules prevents Bim EL from cleavage by caspase-3 in vitro . Jurkat cell lysate was incubated with taxol to assemble microtubules. Recombinant caspase-3 was then added to the reaction. After ultracentrifugation, α-tubulin and Bim were analyzed as in A . ( C ) pBim EL is released from microtubules and cleaved by caspases in apoptotic Jurkat cells. Lysate from Jurkat cells treated with or without staurosporine (Stau) was subjected to the same analysis as in A .( D ) Diagram depicting the activation of a positive feedback apoptotic pathway involving the release of pBim EL from microtubules and caspase cleavage of pBim EL . The cleaved tBim EL (ΔN13) binds more Bcl-2 and has greater apoptotic activity. tBim EL may or may not contain the phosphorylation sites, which have not been mapped in the full-length Bim EL . See the text for details.

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

    Article Title: Caspase cleavage of BimEL triggers a positive feedback amplification of apoptotic signaling

    doi: 10.1073/pnas.0308050100

    Figure Lengend Snippet: pBim EL escapes sequestration by microtubules and becomes accessible to caspase cleavage. ( A ) pBim EL is released from microtubules. Jurkat cell lysate preincubated with or without PAP ( Left ) or lysate from Jurkat cells treated with or without okadaic acid ( Right ) was incubated with taxol and then subjected to ultracentrifugation. α-Tubulin and Bim in the pellet (P), supernatant (S), or lysate before centrifugation (T) were detected by Western blotting. ( B ) The association with microtubules prevents Bim EL from cleavage by caspase-3 in vitro . Jurkat cell lysate was incubated with taxol to assemble microtubules. Recombinant caspase-3 was then added to the reaction. After ultracentrifugation, α-tubulin and Bim were analyzed as in A . ( C ) pBim EL is released from microtubules and cleaved by caspases in apoptotic Jurkat cells. Lysate from Jurkat cells treated with or without staurosporine (Stau) was subjected to the same analysis as in A .( D ) Diagram depicting the activation of a positive feedback apoptotic pathway involving the release of pBim EL from microtubules and caspase cleavage of pBim EL . The cleaved tBim EL (ΔN13) binds more Bcl-2 and has greater apoptotic activity. tBim EL may or may not contain the phosphorylation sites, which have not been mapped in the full-length Bim EL . See the text for details.

    Article Snippet: In vitro cleavage coupled with microtubule assembly was carried out by incubating the cleared Jurkat cell lysate (from 1 × 106 cells) with 5 μg/ml taxol at 37°C for 20 min. After microtubules were assembled, 100 units of recombinant caspase-3 (Calbiochem) were added.

    Techniques: Incubation, Centrifugation, Western Blot, In Vitro, Recombinant, Activation Assay, Activity Assay

    Requirement for an intact BH3 domain on the cytoplasmic face of the mitochondrial outer membrane. ( A ) Sequence of p15 BID BH3 domain mutants mIII.1 and mIII.4. Shaded box indicates the caspase-3 cleavage site DEMD that generates the p11 fragment. ( B ) Treatment of mitochondria with recombinant caspase-3 after targeting wild-type (wt) and caspase-cleavage site mutant p15 BID mIII.1. (Lane 1 ) Amount of p15 BID targeted. (Lane 2 ) Treatment with caspase-3 at 4°C. (Lane 3 ) Treatment with caspase-3 at 30°C. p15 BID and p11 BID are visualized by 35 S fluorography. ( C ) In vitro targeting and cytochrome c release by p15 BID wild type vs mIII.4. (*) A cross-reactive protein present in IVT mix. ( D ) In vitro targeting and cytochrome c release by BIDα345/TM wild-type and mutant mIII.4.

    Journal: Genes & Development

    Article Title: tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c

    doi:

    Figure Lengend Snippet: Requirement for an intact BH3 domain on the cytoplasmic face of the mitochondrial outer membrane. ( A ) Sequence of p15 BID BH3 domain mutants mIII.1 and mIII.4. Shaded box indicates the caspase-3 cleavage site DEMD that generates the p11 fragment. ( B ) Treatment of mitochondria with recombinant caspase-3 after targeting wild-type (wt) and caspase-cleavage site mutant p15 BID mIII.1. (Lane 1 ) Amount of p15 BID targeted. (Lane 2 ) Treatment with caspase-3 at 4°C. (Lane 3 ) Treatment with caspase-3 at 30°C. p15 BID and p11 BID are visualized by 35 S fluorography. ( C ) In vitro targeting and cytochrome c release by p15 BID wild type vs mIII.4. (*) A cross-reactive protein present in IVT mix. ( D ) In vitro targeting and cytochrome c release by BIDα345/TM wild-type and mutant mIII.4.

    Article Snippet: For post-caspase treatment, 0.1 μg of recombinant caspase-3 (Pharmingen) was added following the targeting reaction.

    Techniques: Sequencing, Recombinant, Mutagenesis, In Vitro

    Western blot was utilized to evaluate the levels of pro-caspase-3, cleaved caspase-3 and α -tubulin. ( a ) SFN-Cys downregulated the level of pro-caspase-3. ( b ) SFN-Cys upregulated the expression of cleaved caspase-3 via increased concentrations; ( c ) and ( d ) SFN-Cys decreased α -tubulin, whereas PD98059 reserved this process. ( e ) In vitro , recombinant caspase-3 cleaved α -tubulin to produce a band of 53 kDa, suggesting that SFN-Cys activated caspase-3 might degrade α -tubulin. Each experiment was performed in triplicate. Error bars indicate uncertainty errors in graphs to relay 95% of confidence in the interpreted data. * Indicates P

    Journal: Cell Death Discovery

    Article Title: Sulforaphane-cysteine-induced apoptosis via phosphorylated ERK1/2-mediated maspin pathway in human non-small cell lung cancer cells

    doi: 10.1038/cddiscovery.2017.25

    Figure Lengend Snippet: Western blot was utilized to evaluate the levels of pro-caspase-3, cleaved caspase-3 and α -tubulin. ( a ) SFN-Cys downregulated the level of pro-caspase-3. ( b ) SFN-Cys upregulated the expression of cleaved caspase-3 via increased concentrations; ( c ) and ( d ) SFN-Cys decreased α -tubulin, whereas PD98059 reserved this process. ( e ) In vitro , recombinant caspase-3 cleaved α -tubulin to produce a band of 53 kDa, suggesting that SFN-Cys activated caspase-3 might degrade α -tubulin. Each experiment was performed in triplicate. Error bars indicate uncertainty errors in graphs to relay 95% of confidence in the interpreted data. * Indicates P

    Article Snippet: Recombinant caspase-3 was purchased from Sino Biological Inc. (Beijing, China).

    Techniques: Western Blot, Expressing, In Vitro, Recombinant

    Calpain activation in acute and chronic 3-NP models. A–C , Homogenates prepared from striatum and cerebral cortex of 3-NP-treated and control (CTRL) rats were analyzed by SDS-PAGE followed by Western blot for fodrin (known substrate of calpain and caspase-3) and assayed for calpain-dependent proteolytic activity using the fluorogenic substrate N -succinyl-LY-AMC ( D ). E , Western blot analysis ofμ-calpain cleavage in cytosol fractions from striatum and cerebral cortex of 3-NP-treated animals. The typical calpain-dependent cleavage of fodrin appearing as a 145/150 kDa doublet is found in the striatum of both acute ( A ) and chronic ( B ) 3-NP models (each lane contains pooled samples from 5–8 animals). Note in A the presence between two nonspecific (N.S.) bands of a 120 kDa band (asterisk), possibly because of a caspase-3-mediated fodrin breakdown product. In B , chronic 3-NP animals were analyzed every 6 hr showing that the intensity of this doublet increases on day 4 and is maximal at day 5. Western blots for fodrin in cerebral cortex were slightly overexposed. C shows that the pattern found in the striatum of 3-NP-treated animals (day 5) is clearly different from the caspase-3-mediated cleavage of fodrin observed after in vitro digestion of control samples with recombinant caspase-3 (CTRL + R. Casp-3). Note the prominent increase in the proteolytic activity of calpain and the presence of cleaved/active form ofμ-calpain in the striatum of chronic 3-NP rats, whereas no changes were found within the cerebral cortex of the same animals. Data are means ± SEM determined in 5–10 animals. N.D., Not detectable. ★ p

    Journal: The Journal of Neuroscience

    Article Title: Calpain Is a Major Cell Death Effector in Selective Striatal Degeneration Induced In Vivo by 3-Nitropropionate: Implications for Huntington's Disease

    doi: 10.1523/JNEUROSCI.23-12-05020.2003

    Figure Lengend Snippet: Calpain activation in acute and chronic 3-NP models. A–C , Homogenates prepared from striatum and cerebral cortex of 3-NP-treated and control (CTRL) rats were analyzed by SDS-PAGE followed by Western blot for fodrin (known substrate of calpain and caspase-3) and assayed for calpain-dependent proteolytic activity using the fluorogenic substrate N -succinyl-LY-AMC ( D ). E , Western blot analysis ofμ-calpain cleavage in cytosol fractions from striatum and cerebral cortex of 3-NP-treated animals. The typical calpain-dependent cleavage of fodrin appearing as a 145/150 kDa doublet is found in the striatum of both acute ( A ) and chronic ( B ) 3-NP models (each lane contains pooled samples from 5–8 animals). Note in A the presence between two nonspecific (N.S.) bands of a 120 kDa band (asterisk), possibly because of a caspase-3-mediated fodrin breakdown product. In B , chronic 3-NP animals were analyzed every 6 hr showing that the intensity of this doublet increases on day 4 and is maximal at day 5. Western blots for fodrin in cerebral cortex were slightly overexposed. C shows that the pattern found in the striatum of 3-NP-treated animals (day 5) is clearly different from the caspase-3-mediated cleavage of fodrin observed after in vitro digestion of control samples with recombinant caspase-3 (CTRL + R. Casp-3). Note the prominent increase in the proteolytic activity of calpain and the presence of cleaved/active form ofμ-calpain in the striatum of chronic 3-NP rats, whereas no changes were found within the cerebral cortex of the same animals. Data are means ± SEM determined in 5–10 animals. N.D., Not detectable. ★ p

    Article Snippet: The in vitro proteolysis of endogenous fodrin and huntingtin was studied after incubation (1 hr at 37°C) of control samples (20 μg of protein) with purified μ-calpain (0.2 U; Calbiochem) or recombinant caspase-3 (50 ng; Biomol) in 100 μl of the assay buffer A or B, respectively.

    Techniques: Activation Assay, SDS Page, Western Blot, Activity Assay, In Vitro, Recombinant

    Study of caspase-3 in rats after acute or chronic 3-NP treatment. A , Caspase-3-related DEVDase proteolytic activity in striatal and cortical homogenates. B , Analysis by SDS-PAGE followed by Western blot for the active form of caspase-3. Recombinant caspase-3 (Casp.3) was loaded in the left lane as a positive control. Note that accumulation of the p20 subunit of the active form of caspase-3 (seen as a 17/19 kDa doublet) is detected only in the acute 3-NP model at 48 hr. Ten micrograms of protein were loaded per lane. C , Immunohistochemical analysis of activated caspases-3 showing low levels of immunoreactivity in the striatum in control and chronic model, whereas marked increased striatal labeling in detected in the acute model (arrows). N.D., Not detectable. Data are means ± SEM determined in 5–10 animals. ★ p

    Journal: The Journal of Neuroscience

    Article Title: Calpain Is a Major Cell Death Effector in Selective Striatal Degeneration Induced In Vivo by 3-Nitropropionate: Implications for Huntington's Disease

    doi: 10.1523/JNEUROSCI.23-12-05020.2003

    Figure Lengend Snippet: Study of caspase-3 in rats after acute or chronic 3-NP treatment. A , Caspase-3-related DEVDase proteolytic activity in striatal and cortical homogenates. B , Analysis by SDS-PAGE followed by Western blot for the active form of caspase-3. Recombinant caspase-3 (Casp.3) was loaded in the left lane as a positive control. Note that accumulation of the p20 subunit of the active form of caspase-3 (seen as a 17/19 kDa doublet) is detected only in the acute 3-NP model at 48 hr. Ten micrograms of protein were loaded per lane. C , Immunohistochemical analysis of activated caspases-3 showing low levels of immunoreactivity in the striatum in control and chronic model, whereas marked increased striatal labeling in detected in the acute model (arrows). N.D., Not detectable. Data are means ± SEM determined in 5–10 animals. ★ p

    Article Snippet: The in vitro proteolysis of endogenous fodrin and huntingtin was studied after incubation (1 hr at 37°C) of control samples (20 μg of protein) with purified μ-calpain (0.2 U; Calbiochem) or recombinant caspase-3 (50 ng; Biomol) in 100 μl of the assay buffer A or B, respectively.

    Techniques: Activity Assay, SDS Page, Western Blot, Recombinant, Positive Control, Immunohistochemistry, Labeling

    Spectral response and caspase sensing of CaspeR3 . A. Excitation (dashed lines) and emission (solid lines) spectra for TagGFP (green) and TagRFP (red). Spectral overlap is filled with gray. B. Emission spectra of CaspeR3 before (dashed line) and after digestion by Caspase 3 (solid line). C. Green-to-red emission ratio change of CaspeR3 upon staurosporine-induced apoptosis. Approximately 40–50 min after staurosporine infusion, cells demonstrated pronounced changes fluorescence signal ratio. Emission ratio shown for 5 cells, time point aligned to the median of ratio changes, individual for each cell. Excitation at 488 nm, emission was detected at 500–530 nm and 560–600 nm. D. TagGFP fluorescence lifetime τ φ (solid lines) and τ M (dashed lines) changes for CaspeR3 during staurosporine-induced apoptosis. Excitation was at 488 nm and donor fluorescence emission was passed through a 500–530 nm bandpass filter. E. Two channel fluorescence imaging of CaspeR3 upon staurosporine-induced apoptosis in HeLa cells. Time, in minutes, is shown after staurosporine infusion.

    Journal: BMC Biotechnology

    Article Title: Practical and reliable FRET/FLIM pair of fluorescent proteins

    doi: 10.1186/1472-6750-9-24

    Figure Lengend Snippet: Spectral response and caspase sensing of CaspeR3 . A. Excitation (dashed lines) and emission (solid lines) spectra for TagGFP (green) and TagRFP (red). Spectral overlap is filled with gray. B. Emission spectra of CaspeR3 before (dashed line) and after digestion by Caspase 3 (solid line). C. Green-to-red emission ratio change of CaspeR3 upon staurosporine-induced apoptosis. Approximately 40–50 min after staurosporine infusion, cells demonstrated pronounced changes fluorescence signal ratio. Emission ratio shown for 5 cells, time point aligned to the median of ratio changes, individual for each cell. Excitation at 488 nm, emission was detected at 500–530 nm and 560–600 nm. D. TagGFP fluorescence lifetime τ φ (solid lines) and τ M (dashed lines) changes for CaspeR3 during staurosporine-induced apoptosis. Excitation was at 488 nm and donor fluorescence emission was passed through a 500–530 nm bandpass filter. E. Two channel fluorescence imaging of CaspeR3 upon staurosporine-induced apoptosis in HeLa cells. Time, in minutes, is shown after staurosporine infusion.

    Article Snippet: Direct monitoring of the donor/acceptor emission ratio demonstrated up to 5-fold ratio changes upon cleavage of CaspeR3 by recombinant caspase 3 (BioCat GmbH)in vitro (Fig. and Additional file ).

    Techniques: Fluorescence, Imaging

    δ-Catenin is cleaved by caspase-3 in vitro and in apoptotic extracts. A , biotin-conjugated Xenopus δ-catenin was cleaved by recombinant active caspase-3 in vitro . Intact protein or cleaved fragments (labeled with asterisks ) were visualized

    Journal: The Journal of Biological Chemistry

    Article Title: Caspase-3 Cleavage Links ?-Catenin to the Novel Nuclear Protein ZIFCAT *

    doi: 10.1074/jbc.M110.167544

    Figure Lengend Snippet: δ-Catenin is cleaved by caspase-3 in vitro and in apoptotic extracts. A , biotin-conjugated Xenopus δ-catenin was cleaved by recombinant active caspase-3 in vitro . Intact protein or cleaved fragments (labeled with asterisks ) were visualized

    Article Snippet: Following translation, 5 μl of the reaction mix was incubated with recombinant caspase-3 (BD Pharmingen) at 37 °C for 1 h.

    Techniques: In Vitro, Recombinant, Labeling

    Identification of caspase-3 consensus motif within δ-catenin. A , recombinant Armadillo domain of δ-catenin was cleaved by active caspase-3 in vitro , with the resulting fragments resolved by SDS-PAGE and stained with Coomassie Brilliant

    Journal: The Journal of Biological Chemistry

    Article Title: Caspase-3 Cleavage Links ?-Catenin to the Novel Nuclear Protein ZIFCAT *

    doi: 10.1074/jbc.M110.167544

    Figure Lengend Snippet: Identification of caspase-3 consensus motif within δ-catenin. A , recombinant Armadillo domain of δ-catenin was cleaved by active caspase-3 in vitro , with the resulting fragments resolved by SDS-PAGE and stained with Coomassie Brilliant

    Article Snippet: Following translation, 5 μl of the reaction mix was incubated with recombinant caspase-3 (BD Pharmingen) at 37 °C for 1 h.

    Techniques: Recombinant, In Vitro, SDS Page, Staining

    NS3694 inhibits TNF-induced effector caspase activation and apoptosis in MCF-casp3 cells. MCF-casp3 cells were left untreated (UN) or pretreated with the indicated concentrations of DEVD-CHO or zVAD-fmk for 1 h before the indicated concentrations of TNF were added. NS3694 was added at 10 to 100 μM at the same time as TNF. (A) The survival of the cells was analyzed by the MTT assay after 48 h of TNF treatment. The means ± standard deviations (error bars) of three independent experiments are shown. (B) After 17 h of treatment, the cells were lysed, and the DEVDase activity in lysates was assessed by spectrofluorometric quantification of DEVD-AFC cleavage and presented as picomoles per minute. The means ± standard deviations (error bars) for three samples in a representative experiment are shown. The experiments were repeated several times with similar results.

    Journal: Molecular and Cellular Biology

    Article Title: Diarylurea Compounds Inhibit Caspase Activation by Preventing the Formation of the Active 700-Kilodalton Apoptosome Complex

    doi: 10.1128/MCB.23.21.7829-7837.2003

    Figure Lengend Snippet: NS3694 inhibits TNF-induced effector caspase activation and apoptosis in MCF-casp3 cells. MCF-casp3 cells were left untreated (UN) or pretreated with the indicated concentrations of DEVD-CHO or zVAD-fmk for 1 h before the indicated concentrations of TNF were added. NS3694 was added at 10 to 100 μM at the same time as TNF. (A) The survival of the cells was analyzed by the MTT assay after 48 h of TNF treatment. The means ± standard deviations (error bars) of three independent experiments are shown. (B) After 17 h of treatment, the cells were lysed, and the DEVDase activity in lysates was assessed by spectrofluorometric quantification of DEVD-AFC cleavage and presented as picomoles per minute. The means ± standard deviations (error bars) for three samples in a representative experiment are shown. The experiments were repeated several times with similar results.

    Article Snippet: To test whether NS3694, NS1784, and NS1764 were specific caspase inhibitors, they were incubated with recombinant caspase 3 or caspase 9, and caspase activity was measured using DEVD-AFC and LEHD-AFC as specific substrates for caspase 3 and caspase 9, respectively.

    Techniques: Activation Assay, MTT Assay, Activity Assay

    NS3694 blocks the formation of the ∼700-kDa apoptosome complex. (A) Increasing concentrations of NS3694 were coincubated with THP.1 cell lysates (10 mg) in the presence (+) and absence (−) of 2 mM dATP-MgCl 2 for 30 min at 37°C before assaying for DEVDase activity (essentially caspases 3 and 7) and caspase 9 (Casp-9) and caspase 3 (Casp-3) processing as described in Materials and Methods. The positions of the procaspases (Pro) and processed forms (p35 and p37 [p35/37] and p19 and p17 [p19/p17]) are indicated to the right of the blots. NS3694 was added in a dimethyl sulfoxide solution, and control samples were incubated with equivalent amounts of the solvent, which did not inhibit caspase activation. (B) Two-milligram aliquots of control THP.1 cell lysates and dATP-activated THP.1 cell lysates in the presence and absence of NS3694 (500 μM) were fractionated on Superose 6 columns, and the fractions were analyzed for Apaf-1 and caspase 9 (Casp-9). The sizes of the various peaks were determined by using gel filtration marker proteins, and the elution positions of the ∼700-kDa and ∼1.4-MDa apoptosome complexes and aldolase (158 kDa) are indicated above the blots. Processed caspase 9 was not detected in the control samples and Ns3694-treated samples. In the dATP-activated sample, procaspase 9 (pro) was totally processed to its active subunits. The results shown are from a representative experiment, and similar data were obtained in a repeated experiment. Numbers in boxes, fraction numbers.

    Journal: Molecular and Cellular Biology

    Article Title: Diarylurea Compounds Inhibit Caspase Activation by Preventing the Formation of the Active 700-Kilodalton Apoptosome Complex

    doi: 10.1128/MCB.23.21.7829-7837.2003

    Figure Lengend Snippet: NS3694 blocks the formation of the ∼700-kDa apoptosome complex. (A) Increasing concentrations of NS3694 were coincubated with THP.1 cell lysates (10 mg) in the presence (+) and absence (−) of 2 mM dATP-MgCl 2 for 30 min at 37°C before assaying for DEVDase activity (essentially caspases 3 and 7) and caspase 9 (Casp-9) and caspase 3 (Casp-3) processing as described in Materials and Methods. The positions of the procaspases (Pro) and processed forms (p35 and p37 [p35/37] and p19 and p17 [p19/p17]) are indicated to the right of the blots. NS3694 was added in a dimethyl sulfoxide solution, and control samples were incubated with equivalent amounts of the solvent, which did not inhibit caspase activation. (B) Two-milligram aliquots of control THP.1 cell lysates and dATP-activated THP.1 cell lysates in the presence and absence of NS3694 (500 μM) were fractionated on Superose 6 columns, and the fractions were analyzed for Apaf-1 and caspase 9 (Casp-9). The sizes of the various peaks were determined by using gel filtration marker proteins, and the elution positions of the ∼700-kDa and ∼1.4-MDa apoptosome complexes and aldolase (158 kDa) are indicated above the blots. Processed caspase 9 was not detected in the control samples and Ns3694-treated samples. In the dATP-activated sample, procaspase 9 (pro) was totally processed to its active subunits. The results shown are from a representative experiment, and similar data were obtained in a repeated experiment. Numbers in boxes, fraction numbers.

    Article Snippet: To test whether NS3694, NS1784, and NS1764 were specific caspase inhibitors, they were incubated with recombinant caspase 3 or caspase 9, and caspase activity was measured using DEVD-AFC and LEHD-AFC as specific substrates for caspase 3 and caspase 9, respectively.

    Techniques: Activity Assay, Incubation, Activation Assay, Filtration, Marker, Multiple Displacement Amplification

    Diarylurea compounds do not inhibit the activity of caspase 9 or caspase 3. The enzymatic activity of recombinant caspase 9 (rCaspase-9) (A) and recombinant caspase 3 (rCaspase-3) (B) was assessed by spectrofluorometric quantification of LEHD-AFC or DEVD-AFC cleavage, respectively, in the presence (+) or absence of 100 μM NS3694, NS1784, NS1764, DEVD-CHO (DEVD), or zVAD-fmk (zVAD). Enzymatic activity is presented as picomoles per minute, and the means ± standard deviations (error bars) for three samples are shown. Data are representative of the results from three independent assays.

    Journal: Molecular and Cellular Biology

    Article Title: Diarylurea Compounds Inhibit Caspase Activation by Preventing the Formation of the Active 700-Kilodalton Apoptosome Complex

    doi: 10.1128/MCB.23.21.7829-7837.2003

    Figure Lengend Snippet: Diarylurea compounds do not inhibit the activity of caspase 9 or caspase 3. The enzymatic activity of recombinant caspase 9 (rCaspase-9) (A) and recombinant caspase 3 (rCaspase-3) (B) was assessed by spectrofluorometric quantification of LEHD-AFC or DEVD-AFC cleavage, respectively, in the presence (+) or absence of 100 μM NS3694, NS1784, NS1764, DEVD-CHO (DEVD), or zVAD-fmk (zVAD). Enzymatic activity is presented as picomoles per minute, and the means ± standard deviations (error bars) for three samples are shown. Data are representative of the results from three independent assays.

    Article Snippet: To test whether NS3694, NS1784, and NS1764 were specific caspase inhibitors, they were incubated with recombinant caspase 3 or caspase 9, and caspase activity was measured using DEVD-AFC and LEHD-AFC as specific substrates for caspase 3 and caspase 9, respectively.

    Techniques: Activity Assay, Recombinant

    Identification of diarylurea compounds as inhibitors of the apoptosome complex. (A) Cytosolic extract prepared from HeLa cells was left untreated (UN) or incubated with cytochrome  c  (Cc; 1 μM) and dATP (1 mM) in the presence (+) or absence of 100 μM concentrations of library compounds, 1 μM zVAD-fmk (zVAD), or 0.1 μM DEVD-CHO (DEVD). Caspase 3-like activity was assessed by spectrofluorometric quantification of DEVD-AFC cleavage as described in Materials and Methods. Percent inhibition is indicated above the bars. The results of three of the most potent compounds (NS1764, NS1784, and NS3694) are presented. (B) Chemical structures of NS1764, NS1784, and NS3694.

    Journal: Molecular and Cellular Biology

    Article Title: Diarylurea Compounds Inhibit Caspase Activation by Preventing the Formation of the Active 700-Kilodalton Apoptosome Complex

    doi: 10.1128/MCB.23.21.7829-7837.2003

    Figure Lengend Snippet: Identification of diarylurea compounds as inhibitors of the apoptosome complex. (A) Cytosolic extract prepared from HeLa cells was left untreated (UN) or incubated with cytochrome c (Cc; 1 μM) and dATP (1 mM) in the presence (+) or absence of 100 μM concentrations of library compounds, 1 μM zVAD-fmk (zVAD), or 0.1 μM DEVD-CHO (DEVD). Caspase 3-like activity was assessed by spectrofluorometric quantification of DEVD-AFC cleavage as described in Materials and Methods. Percent inhibition is indicated above the bars. The results of three of the most potent compounds (NS1764, NS1784, and NS3694) are presented. (B) Chemical structures of NS1764, NS1784, and NS3694.

    Article Snippet: To test whether NS3694, NS1784, and NS1764 were specific caspase inhibitors, they were incubated with recombinant caspase 3 or caspase 9, and caspase activity was measured using DEVD-AFC and LEHD-AFC as specific substrates for caspase 3 and caspase 9, respectively.

    Techniques: Incubation, Activity Assay, Inhibition

    NS3694 inhibits cytochrome  c - and dATP-induced DEVDase activation and processing of caspases and caspase substrates. Cytosolic extracts prepared from HeLa cells were left untreated (UN) or incubated at 37°C for 120 min with cytochrome  c  (Cc; 1 μM) and dATP (1 mM) in the presence (+) or absence of NS3694 (10 to 100 μM), DEVD-CHO (0.1 μM), and zVAD-fmk (1 μM). Caspase 3-like activity was assessed by spectrofluorometric quantification of DEVD-AFC cleavage and presented as picomoles per minute. After measurement of the DEVDase activity, the same samples were analyzed by immunoblotting using antibodies against the indicated proteins. The means ± standard deviations (error bars) of three independent experiments are shown, and the immunoblot analysis was repeated twice with essentially identical results.

    Journal: Molecular and Cellular Biology

    Article Title: Diarylurea Compounds Inhibit Caspase Activation by Preventing the Formation of the Active 700-Kilodalton Apoptosome Complex

    doi: 10.1128/MCB.23.21.7829-7837.2003

    Figure Lengend Snippet: NS3694 inhibits cytochrome c - and dATP-induced DEVDase activation and processing of caspases and caspase substrates. Cytosolic extracts prepared from HeLa cells were left untreated (UN) or incubated at 37°C for 120 min with cytochrome c (Cc; 1 μM) and dATP (1 mM) in the presence (+) or absence of NS3694 (10 to 100 μM), DEVD-CHO (0.1 μM), and zVAD-fmk (1 μM). Caspase 3-like activity was assessed by spectrofluorometric quantification of DEVD-AFC cleavage and presented as picomoles per minute. After measurement of the DEVDase activity, the same samples were analyzed by immunoblotting using antibodies against the indicated proteins. The means ± standard deviations (error bars) of three independent experiments are shown, and the immunoblot analysis was repeated twice with essentially identical results.

    Article Snippet: To test whether NS3694, NS1784, and NS1764 were specific caspase inhibitors, they were incubated with recombinant caspase 3 or caspase 9, and caspase activity was measured using DEVD-AFC and LEHD-AFC as specific substrates for caspase 3 and caspase 9, respectively.

    Techniques: Activation Assay, Incubation, Activity Assay

    Toxic h-IAPP lag phase intermediates induce β-cell apoptosis, but freshly dissolved h-IAPP (time-zero) and amyloid fibrils do not. Cleaved caspase-3 colorimetric assays of β-cells treated with exogenous h-IAPP lag phase intermediates (blue) show an increase in cleaved caspase-3, but time-zero h-IAPP species (black), h-IAPP amyloid fibrils (red), r-IAPP (green) and buffer (gold) do not. IAPP kinetic species were produced at 25°C in solutions containing 20 μM peptide. Final peptide concentration after dilution into β-cell assays was 14 μM. Data represent mean ± SEM of three replicates per condition. DOI: http://dx.doi.org/10.7554/eLife.12977.008

    Journal: eLife

    Article Title: Time-resolved studies define the nature of toxic IAPP intermediates, providing insight for anti-amyloidosis therapeutics

    doi: 10.7554/eLife.12977

    Figure Lengend Snippet: Toxic h-IAPP lag phase intermediates induce β-cell apoptosis, but freshly dissolved h-IAPP (time-zero) and amyloid fibrils do not. Cleaved caspase-3 colorimetric assays of β-cells treated with exogenous h-IAPP lag phase intermediates (blue) show an increase in cleaved caspase-3, but time-zero h-IAPP species (black), h-IAPP amyloid fibrils (red), r-IAPP (green) and buffer (gold) do not. IAPP kinetic species were produced at 25°C in solutions containing 20 μM peptide. Final peptide concentration after dilution into β-cell assays was 14 μM. Data represent mean ± SEM of three replicates per condition. DOI: http://dx.doi.org/10.7554/eLife.12977.008

    Article Snippet: Recombinant caspase-3 enzyme (R & D Systems) was used as a positive control.

    Techniques: Produced, Concentration Assay

    Cleavage of BeAn virions in vitro by caspase-3 produces 13- and 17-kDa peptides and an increased physical particle/PFU ratio.

    Journal: Journal of Virology

    Article Title: During Infection, Theiler's Virions Are Cleaved by Caspases and Disassembled into Pentamers

    doi: 10.1128/JVI.03035-15

    Figure Lengend Snippet: Cleavage of BeAn virions in vitro by caspase-3 produces 13- and 17-kDa peptides and an increased physical particle/PFU ratio.

    Article Snippet: To further test for proteolysis by caspase-3, BeAn virus purified from infected BHK-21 or M1-D cells was incubated with cytosolic extracts of infected M1-D cells or 10 U of active recombinant caspase-3, and the physical particle/PFU ratios were determined.

    Techniques: In Vitro

    Protein expression of cleaved caspase-3. Western blots for cleaved caspase-3 in naïve, sham, hypoxia–ischemia injured+hypothermia (HI+HypoT), HI+rapid rewarming (HI+rapid RW), and HI+slow rewarming

    Journal: Journal of Cerebral Blood Flow & Metabolism

    Article Title: Rewarming from therapeutic hypothermia induces cortical neuron apoptosis in a swine model of neonatal hypoxic–ischemic encephalopathy

    doi: 10.1038/jcbfm.2014.245

    Figure Lengend Snippet: Protein expression of cleaved caspase-3. Western blots for cleaved caspase-3 in naïve, sham, hypoxia–ischemia injured+hypothermia (HI+HypoT), HI+rapid rewarming (HI+rapid RW), and HI+slow rewarming

    Article Snippet: In some experiments, recombinant active caspase-3 (Biovision, Milipitas, CA, USA) was loaded as a positive control.

    Techniques: Expressing, Western Blot

    The piglets underwent hypoxia–ischemia followed by hypothermia and rapid rewarming. In comparison to artificial CSF (aCSF), subdural administration of caspase-3 inhibitor decreased the number of ( A ) apoptotic profiles by hematoxylin and eosin

    Journal: Journal of Cerebral Blood Flow & Metabolism

    Article Title: Rewarming from therapeutic hypothermia induces cortical neuron apoptosis in a swine model of neonatal hypoxic–ischemic encephalopathy

    doi: 10.1038/jcbfm.2014.245

    Figure Lengend Snippet: The piglets underwent hypoxia–ischemia followed by hypothermia and rapid rewarming. In comparison to artificial CSF (aCSF), subdural administration of caspase-3 inhibitor decreased the number of ( A ) apoptotic profiles by hematoxylin and eosin

    Article Snippet: In some experiments, recombinant active caspase-3 (Biovision, Milipitas, CA, USA) was loaded as a positive control.

    Techniques:

    SASH1 overexpression induces apoptosis. ( a ) HeLa cells were transfected with Flag-SASH1 (OE=overexpressed) and irradiated with ultraviolet light C (UVC) 3 h to induce apoptosis (10–50 mJ/cm 2 ). Quantification of PARP1, cleaved caspase-3 and cleaved caspase-9 levels indicated below blots wasperformed with ImageJ (University of Wisconsin, Madison, USA). Statistical analysis was performed with Student's T -test with * P

    Journal: Cell Death & Disease

    Article Title: Activation and cleavage of SASH1 by caspase-3 mediates an apoptotic response

    doi: 10.1038/cddis.2016.364

    Figure Lengend Snippet: SASH1 overexpression induces apoptosis. ( a ) HeLa cells were transfected with Flag-SASH1 (OE=overexpressed) and irradiated with ultraviolet light C (UVC) 3 h to induce apoptosis (10–50 mJ/cm 2 ). Quantification of PARP1, cleaved caspase-3 and cleaved caspase-9 levels indicated below blots wasperformed with ImageJ (University of Wisconsin, Madison, USA). Statistical analysis was performed with Student's T -test with * P

    Article Snippet: SASH1-bound beads were incubated for 16 h in protease inhibitor-free buffer (25 mM HEPES, 0.1% (w/v) CHAPS, 10 mM DTT, pH 7.5) with and without 2 U of active recombinant caspase-3 (Abcam, Melbourne, VIC, Australia).

    Techniques: Over Expression, Transfection, Irradiation

    SASH1 is cleaved by caspase-3 at D230. ( a ) Immunoblot of U2OS cells overexpressing N- or C-terminal Flag-tagged SASH1. ( b ) Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel containing immunoprecipitation of C-terminal Flag-tagged SASH1. ( c ) Schematic diagram of SASH1 protein domains indicating cleavage site and N-terminal sequencing of amino acids (GSYPTF), which are preceded by a caspase-3 recognition site (DXXD). ( d ) SASH1 was immunoprecipitated from SASH1-Flag stably expressing U2OS cells using M2 Flag beads, followed by induction of apoptosis by UVC (50 mJ/cm 2 , 3 h) with cells pre-treated (30 min) with caspase-3 inhibitor Z-VAD-FMK (20 μ M). ( e ) SASH1 is cleaved by recombinant caspase-3. SASH1 was immunoprecipitated from U2OS cells as per ( d ) and incubated with recombinant caspase-3 (2 U, 16 h). ( f ) Ectopically expressed 230E mutant SASH1 is not cleaved following UVC exposure. HeLa cells overexpressing wild-type or D230E were treated with UVC (50 mJ/cm 2 , 3 h). Cell extracts were immunoblotted and incubated with the indicated antibodies. ( g ) SASH1 230E mutant is not cleaved by recombinant caspase-3. Cell extracts taken from HeLa cells overexpressing wild-type and 230E SASH1 were incubated with recombinant caspase-3 (2 U, 16 h)

    Journal: Cell Death & Disease

    Article Title: Activation and cleavage of SASH1 by caspase-3 mediates an apoptotic response

    doi: 10.1038/cddis.2016.364

    Figure Lengend Snippet: SASH1 is cleaved by caspase-3 at D230. ( a ) Immunoblot of U2OS cells overexpressing N- or C-terminal Flag-tagged SASH1. ( b ) Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel containing immunoprecipitation of C-terminal Flag-tagged SASH1. ( c ) Schematic diagram of SASH1 protein domains indicating cleavage site and N-terminal sequencing of amino acids (GSYPTF), which are preceded by a caspase-3 recognition site (DXXD). ( d ) SASH1 was immunoprecipitated from SASH1-Flag stably expressing U2OS cells using M2 Flag beads, followed by induction of apoptosis by UVC (50 mJ/cm 2 , 3 h) with cells pre-treated (30 min) with caspase-3 inhibitor Z-VAD-FMK (20 μ M). ( e ) SASH1 is cleaved by recombinant caspase-3. SASH1 was immunoprecipitated from U2OS cells as per ( d ) and incubated with recombinant caspase-3 (2 U, 16 h). ( f ) Ectopically expressed 230E mutant SASH1 is not cleaved following UVC exposure. HeLa cells overexpressing wild-type or D230E were treated with UVC (50 mJ/cm 2 , 3 h). Cell extracts were immunoblotted and incubated with the indicated antibodies. ( g ) SASH1 230E mutant is not cleaved by recombinant caspase-3. Cell extracts taken from HeLa cells overexpressing wild-type and 230E SASH1 were incubated with recombinant caspase-3 (2 U, 16 h)

    Article Snippet: SASH1-bound beads were incubated for 16 h in protease inhibitor-free buffer (25 mM HEPES, 0.1% (w/v) CHAPS, 10 mM DTT, pH 7.5) with and without 2 U of active recombinant caspase-3 (Abcam, Melbourne, VIC, Australia).

    Techniques: Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Immunoprecipitation, Sequencing, Stable Transfection, Expressing, Recombinant, Incubation, Mutagenesis

    Design and pharmacological evaluation of TRP601. ( a ) Substrate binding region of caspase-2 (Casp2) in complex with TRP601. The quinolin-2-carbonyl-Val-Asp(OMe)-Val-Ala-Asp(OMe)-CH 2 - moiety is shown in sticks representation with the atoms represented as follows: gray=carbon; white=hydrogen; blue=nitrogen; and red=oxygen. The S–C covalent bond between the Cys-155 residue and the C terminus CH 2 of TRP601 is represented in yellow. The electric interactions (hydrogen bonds and salts bridges) are represented by the dashed, green lines. The enzyme residues that interact with the inhibitor are shown by the wireframe representation. Right panel: the table shows the minimal energy (kcal/mol, E min ) of the Casp2–TRP601 complex resulting from electric or Coulombic component ( E c ) together with repulsion–attraction component ( E vw ). The interaction energy of the Casp2–TRP601 complex is −147.28 kcal/mol. Owing to the C–S covalent bond, the Asp residue in P1 contributes to about 30% of the interaction energy. The Asp in P4 and the Val in P5 have important non-covalent contributions to stabilization of the complex, 21% and 17%, respectively. Each of the other two residues (Ala in P2 and Val in P3) contributes to about 12% of the interaction energy. ( b ) Structure of TRP601. ( c ) Representative dose–response curve of human recombinant Casp2 and Casp3 inhibition by TRP601. Initial velocities were determined from standard colorimetric microplate assays. ( d ) Kinetic off rate (k 3 / K i ) parameters of irreversible caspase inhibitors on Casp2 and Casp3. ( e ) TRP601 inhibits neuronal caspase activities and prevents serum deprivation (SD)-induced cell death. High-density E14 cortical neuron cultures were subjected to 24 h SD in the presence or absence of 50 μM TRP601. Histograms indicate the means (±S.D.) of 15 independent experiments. ( f ) Representative pharmacokinetic of TRP601 after intravenous (i.v.) administration in adult rats, through liquid chromatography-mass spectrometry (LC-MS/MS) detection in the plasma and brain homogenates. Note that following intraperitoneal (i.p.) administration of the same dose, TRP601 was detected in the brain at 0.25 h (brain C max =25 ng/ml) and the C max (20 ng/ml) was obtained in the plasma according to a plateau between 0.5 and 2 h. ( g – j ) TRP601 reduces excitotoxic lesions in neonates. The 5-day-old mice were subjected to intracerebral ibotenate injection and killed at different time points ( g =5 days; h and i =24 h; j =8 h) following the excitotoxic challenge to determine the impact of TRP601, TRP801, and TRP901 (1 mg/kg; i.p.) on lesion severity ( g ), microgliosis ( h ), astrogliosis ( i ), and group II caspases activity ( j ). Histograms show mean lesion volume ( g ; ▪, vehicle, n =16; TRP601, n =16; TRP801, n =7; TRP901, n =8), cell density ( h and i ; n =10 per group), or VDVADase activity ( j ; n =5 per group)±S.E.M. Asterisks indicate differences from control (▪) ( * P

    Journal: Cell Death & Disease

    Article Title: Targeting neonatal ischemic brain injury with a pentapeptide-based irreversible caspase inhibitor

    doi: 10.1038/cddis.2011.87

    Figure Lengend Snippet: Design and pharmacological evaluation of TRP601. ( a ) Substrate binding region of caspase-2 (Casp2) in complex with TRP601. The quinolin-2-carbonyl-Val-Asp(OMe)-Val-Ala-Asp(OMe)-CH 2 - moiety is shown in sticks representation with the atoms represented as follows: gray=carbon; white=hydrogen; blue=nitrogen; and red=oxygen. The S–C covalent bond between the Cys-155 residue and the C terminus CH 2 of TRP601 is represented in yellow. The electric interactions (hydrogen bonds and salts bridges) are represented by the dashed, green lines. The enzyme residues that interact with the inhibitor are shown by the wireframe representation. Right panel: the table shows the minimal energy (kcal/mol, E min ) of the Casp2–TRP601 complex resulting from electric or Coulombic component ( E c ) together with repulsion–attraction component ( E vw ). The interaction energy of the Casp2–TRP601 complex is −147.28 kcal/mol. Owing to the C–S covalent bond, the Asp residue in P1 contributes to about 30% of the interaction energy. The Asp in P4 and the Val in P5 have important non-covalent contributions to stabilization of the complex, 21% and 17%, respectively. Each of the other two residues (Ala in P2 and Val in P3) contributes to about 12% of the interaction energy. ( b ) Structure of TRP601. ( c ) Representative dose–response curve of human recombinant Casp2 and Casp3 inhibition by TRP601. Initial velocities were determined from standard colorimetric microplate assays. ( d ) Kinetic off rate (k 3 / K i ) parameters of irreversible caspase inhibitors on Casp2 and Casp3. ( e ) TRP601 inhibits neuronal caspase activities and prevents serum deprivation (SD)-induced cell death. High-density E14 cortical neuron cultures were subjected to 24 h SD in the presence or absence of 50 μM TRP601. Histograms indicate the means (±S.D.) of 15 independent experiments. ( f ) Representative pharmacokinetic of TRP601 after intravenous (i.v.) administration in adult rats, through liquid chromatography-mass spectrometry (LC-MS/MS) detection in the plasma and brain homogenates. Note that following intraperitoneal (i.p.) administration of the same dose, TRP601 was detected in the brain at 0.25 h (brain C max =25 ng/ml) and the C max (20 ng/ml) was obtained in the plasma according to a plateau between 0.5 and 2 h. ( g – j ) TRP601 reduces excitotoxic lesions in neonates. The 5-day-old mice were subjected to intracerebral ibotenate injection and killed at different time points ( g =5 days; h and i =24 h; j =8 h) following the excitotoxic challenge to determine the impact of TRP601, TRP801, and TRP901 (1 mg/kg; i.p.) on lesion severity ( g ), microgliosis ( h ), astrogliosis ( i ), and group II caspases activity ( j ). Histograms show mean lesion volume ( g ; ▪, vehicle, n =16; TRP601, n =16; TRP801, n =7; TRP901, n =8), cell density ( h and i ; n =10 per group), or VDVADase activity ( j ; n =5 per group)±S.E.M. Asterisks indicate differences from control (▪) ( * P

    Article Snippet: Assays of chromogenic substrate cleavage contained in 100 μ l caspase buffer (20 mM HEPES, pH 7.4, 0.1% CHAPS, 5 m DTT, 2 m EDTA), 117 ng of human active recombinant caspase-2 (Biomol, Plymouth, PA, USA) or 20 ng of human active recombinant caspase-3 (Sigma; no. C5974), and 100 μ of chromogenic substrate (Ac–DEVD–para -nitroaniline (p NA) for Casp3 and Ac–VDVAD–p NA for Casp2) in the presence of a range of concentrations of inhibitors.

    Techniques: Binding Assay, Recombinant, Inhibition, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Mouse Assay, Injection, Activity Assay

    When neither CTSB deletion nor pH neutralization affected necrosis, we investigated caspase 3 activation and apoptosis at the time point of maximal protease activation in acini following CCK stimulation (30 min). A , pH neutralization with chloroquine

    Journal: The Journal of Biological Chemistry

    Article Title: Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis *

    doi: 10.1074/jbc.M116.718999

    Figure Lengend Snippet: When neither CTSB deletion nor pH neutralization affected necrosis, we investigated caspase 3 activation and apoptosis at the time point of maximal protease activation in acini following CCK stimulation (30 min). A , pH neutralization with chloroquine

    Article Snippet: Nafamostat, a trypsin inhibitor, recombinant human caspase 3, as well as chloroquine were from Sigma.

    Techniques: Neutralization, Activation Assay

    A–C , to study whether trypsin-induced caspase 3 activation translates into apoptosis in vivo , we used two models of experimental pancreatitis and measured apoptosis by TUNEL assay in tissue sections. After 8 h of caerulein-induced pancreatitis,

    Journal: The Journal of Biological Chemistry

    Article Title: Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis *

    doi: 10.1074/jbc.M116.718999

    Figure Lengend Snippet: A–C , to study whether trypsin-induced caspase 3 activation translates into apoptosis in vivo , we used two models of experimental pancreatitis and measured apoptosis by TUNEL assay in tissue sections. After 8 h of caerulein-induced pancreatitis,

    Article Snippet: Nafamostat, a trypsin inhibitor, recombinant human caspase 3, as well as chloroquine were from Sigma.

    Techniques: Activation Assay, In Vivo, TUNEL Assay

    Apoptosis in experimental fetal bladders. A–C is the same field from an obstructed muscle bundle visualized in appropriate wavelengths for propidium iodide ( A ) and TUNEL-FITC ( B ), with C representing the merged image. Five TUNEL-labeled nuclei are indicated ( arrows ). In this field, the numbers of apoptotic nuclei were higher than average; these images were simply used to illustrate the TUNEL technique. Bars , 10 μm. D: The Apoptotic Index in sham ( closed bars , n = 6) and obstructed ( open bars , n = 6) bladders in detrusor SMC, lamina propria cells, and urothelial cells. Note the significant increase of death in obstructed versus sham detrusor SMC and lamina propria cells. E: Increased caspase-3 enzyme activity in full thickness samples from obstructed bladders ( closed bars , n = 5) versus sham bladders ( open bars , n = 5).

    Journal: The American Journal of Pathology

    Article Title: Urinary Outflow Obstruction Increases Apoptosis and Deregulates Bcl-2 and Bax Expression in the Fetal Ovine Bladder

    doi:

    Figure Lengend Snippet: Apoptosis in experimental fetal bladders. A–C is the same field from an obstructed muscle bundle visualized in appropriate wavelengths for propidium iodide ( A ) and TUNEL-FITC ( B ), with C representing the merged image. Five TUNEL-labeled nuclei are indicated ( arrows ). In this field, the numbers of apoptotic nuclei were higher than average; these images were simply used to illustrate the TUNEL technique. Bars , 10 μm. D: The Apoptotic Index in sham ( closed bars , n = 6) and obstructed ( open bars , n = 6) bladders in detrusor SMC, lamina propria cells, and urothelial cells. Note the significant increase of death in obstructed versus sham detrusor SMC and lamina propria cells. E: Increased caspase-3 enzyme activity in full thickness samples from obstructed bladders ( closed bars , n = 5) versus sham bladders ( open bars , n = 5).

    Article Snippet: Absorbency readings were also determined for a p NA standard curve and for quantitative purposes, decreasing dilutions of recombinant active caspase-3 (Chemicon) was also tested by the Assay Kit.

    Techniques: TUNEL Assay, Labeling, Activity Assay

    Histology and cellular turnover in the developing fetal bladder. A–C depict Masson’s trichrome-stained sections at 75 ( A ), 105 ( B ) and 145 ( C ) days gestation, representative of 2, 11 and 6 fetal bladders, respectively. Note increasing thickness of the fetal bladder through gestation. Bars , 400 μm. D: Western blot for PCNA (36 kd) and β-actin (42 kd) of full thickness bladders; note the trend to down-regulation of PCNA expression through gestation into adulthood. This impression was confirmed by PCNA/β-actin ratios. F: A similar decrease in activated caspase-3 activity was noted between 75 and 145 days gestation. In E and F , each dot represents a single bladder sample.

    Journal: The American Journal of Pathology

    Article Title: Urinary Outflow Obstruction Increases Apoptosis and Deregulates Bcl-2 and Bax Expression in the Fetal Ovine Bladder

    doi:

    Figure Lengend Snippet: Histology and cellular turnover in the developing fetal bladder. A–C depict Masson’s trichrome-stained sections at 75 ( A ), 105 ( B ) and 145 ( C ) days gestation, representative of 2, 11 and 6 fetal bladders, respectively. Note increasing thickness of the fetal bladder through gestation. Bars , 400 μm. D: Western blot for PCNA (36 kd) and β-actin (42 kd) of full thickness bladders; note the trend to down-regulation of PCNA expression through gestation into adulthood. This impression was confirmed by PCNA/β-actin ratios. F: A similar decrease in activated caspase-3 activity was noted between 75 and 145 days gestation. In E and F , each dot represents a single bladder sample.

    Article Snippet: Absorbency readings were also determined for a p NA standard curve and for quantitative purposes, decreasing dilutions of recombinant active caspase-3 (Chemicon) was also tested by the Assay Kit.

    Techniques: Staining, Western Blot, Expressing, Activity Assay

    CPT causes in cortical neurons inactivation of Topo-I, accumulation of DNA-SSBs and -DSBs in neurons, and induces caspase 3–dependent apoptosis. ( A ) Hoechst 33258 staining of DIV5 neuron nuclei in vehicle control and 10 μM CPT-treated

    Journal:

    Article Title: Molecular Regulation of DNA Damage-Induced Apoptosis in Neurons of Cerebral Cortex

    doi: 10.1093/cercor/bhn167

    Figure Lengend Snippet: CPT causes in cortical neurons inactivation of Topo-I, accumulation of DNA-SSBs and -DSBs in neurons, and induces caspase 3–dependent apoptosis. ( A ) Hoechst 33258 staining of DIV5 neuron nuclei in vehicle control and 10 μM CPT-treated

    Article Snippet: Human recombinant active caspase-3 (Chemicon) was used as a positive control.

    Techniques: Cycling Probe Technology, Staining

    Characterization of transcriptional changes induced by HDACi and mercurials. a Differentiating cells were treated for 6 days by toxicants (four samples per compound; as in Fig. 1 ) before RNA was prepared and gene expression was measured on Affymetrix microarrays. The 50 genes with highest variance between all samples were selected for clustering (=clustering set). Then, all samples were clustered (Euclidean distance) on the basis of gene expression values for this set. The results are represented as heatmap with each row representing one gene, and the colour of each square indicating the absolute gene expression level ( blue low; green middle; yellow high). b Number of differentially expressed genes (DEG) after exposure to toxicants compared to untreated controls (detailed data are shown in supplemental material). c Recombinant active caspase-3 was incubated for 30 min with respective mercurials at indicated concentrations. Then, the enzymatic activity was determined by a fluorometric assay. The caspase activity is represented in percentage relative to untreated control enzyme. The BMC of the respective mercurial (used in this study for microarray analysis) is indicated by a red line ; data are mean ± SEM; n = 3; panobino, panobinostat (colour figure online)

    Journal: Archives of Toxicology

    Article Title: A transcriptome-based classifier to identify developmental toxicants by stem cell testing: design, validation and optimization for histone deacetylase inhibitors

    doi: 10.1007/s00204-015-1573-y

    Figure Lengend Snippet: Characterization of transcriptional changes induced by HDACi and mercurials. a Differentiating cells were treated for 6 days by toxicants (four samples per compound; as in Fig. 1 ) before RNA was prepared and gene expression was measured on Affymetrix microarrays. The 50 genes with highest variance between all samples were selected for clustering (=clustering set). Then, all samples were clustered (Euclidean distance) on the basis of gene expression values for this set. The results are represented as heatmap with each row representing one gene, and the colour of each square indicating the absolute gene expression level ( blue low; green middle; yellow high). b Number of differentially expressed genes (DEG) after exposure to toxicants compared to untreated controls (detailed data are shown in supplemental material). c Recombinant active caspase-3 was incubated for 30 min with respective mercurials at indicated concentrations. Then, the enzymatic activity was determined by a fluorometric assay. The caspase activity is represented in percentage relative to untreated control enzyme. The BMC of the respective mercurial (used in this study for microarray analysis) is indicated by a red line ; data are mean ± SEM; n = 3; panobino, panobinostat (colour figure online)

    Article Snippet: Caspase-3 inhibition assay by mercurials Recombinant human caspase-3 (Millipore; CC-119) (0.25 U/200 µl reaction volume) in 50 mM Hepes, pH 7.4 containing 1 % sucrose and 0.1 % Chaps was treated with the respective mercurials at 37 °C for 20 min. Caspase-3 activity was then determined by the addition of the substrate N -acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethyl coumarin (NAc-DEVD-afc) (50 µM).

    Techniques: Expressing, Recombinant, Incubation, Activity Assay, Microarray

    Prioritization of FB -modified VAD-FMK peptide caspase inhibitor Chemical structures for R1 substitution of VAD-FMK peptide inhibitor scaffold (A). FB-VAD-FMK (green capped sticks) shown docked into the caspase-3 protein structure with covalent inhibitor removed (PDB ID 3KJF); covalent inhibitor protein attachment at Cys163 is shown in yellow and white VDW spheres. Atom colors: oxygen = red; nitrogen = blue; carbon = grey. Grey VDW surfaces indicate the space-filling shape of the covalent bound inhibitor in PDB ID 3KJF (B). Mean IC 50 values (n ≥ 3) of inhibition of human recombinant caspase-3 by VAD-FMK and analogues as assessed using a luminescence biochemistry assay-based method (Conc = concentration) (C). Caspase-3/7 inhibition with [ 19 F]FB-VAD-FMK (D) or VAD-FMK (E) in untreated or cetuximab-treated DiFi cells (n = 4).

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: A Peptide-Based Positron Emission Tomography Probe for In Vivo Detection of Caspase Activity in Apoptotic Cells

    doi: 10.1158/1078-0432.CCR-13-2444

    Figure Lengend Snippet: Prioritization of FB -modified VAD-FMK peptide caspase inhibitor Chemical structures for R1 substitution of VAD-FMK peptide inhibitor scaffold (A). FB-VAD-FMK (green capped sticks) shown docked into the caspase-3 protein structure with covalent inhibitor removed (PDB ID 3KJF); covalent inhibitor protein attachment at Cys163 is shown in yellow and white VDW spheres. Atom colors: oxygen = red; nitrogen = blue; carbon = grey. Grey VDW surfaces indicate the space-filling shape of the covalent bound inhibitor in PDB ID 3KJF (B). Mean IC 50 values (n ≥ 3) of inhibition of human recombinant caspase-3 by VAD-FMK and analogues as assessed using a luminescence biochemistry assay-based method (Conc = concentration) (C). Caspase-3/7 inhibition with [ 19 F]FB-VAD-FMK (D) or VAD-FMK (E) in untreated or cetuximab-treated DiFi cells (n = 4).

    Article Snippet: Caspase inhibitor dissolved in DMSO (0, 0.1, 1, 10, 100, 1000, or 10000 nM) was combined with recombinant human caspase-3 enzyme (C1224-10UG, Sigma) (100 nM) in 1× phosphate buffered saline (PBS) in microcentrifuge tubes, vortexed, and incubated at 37°C for 30 min. After incubation, Caspase-Glo 3/7 reagent (Promega) was added in accordance with the manufacturer’s instructions.

    Techniques: Modification, Inhibition, Recombinant, Concentration Assay

    AZD-1152-HQPA in vitro exposure results in cell death in DLD-1 and SW620 cell lines Cell cycle analysis by PI flow cytometry (A), caspase-3/7 activity (n = 5) (B), and western blot analysis of cleaved PARP and cleaved caspase-3 (C) 24 h post drug administration. Drug concentrations for western blotting were: 0, 10, 100, 500, 1000, or 5000 nM.

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: A Peptide-Based Positron Emission Tomography Probe for In Vivo Detection of Caspase Activity in Apoptotic Cells

    doi: 10.1158/1078-0432.CCR-13-2444

    Figure Lengend Snippet: AZD-1152-HQPA in vitro exposure results in cell death in DLD-1 and SW620 cell lines Cell cycle analysis by PI flow cytometry (A), caspase-3/7 activity (n = 5) (B), and western blot analysis of cleaved PARP and cleaved caspase-3 (C) 24 h post drug administration. Drug concentrations for western blotting were: 0, 10, 100, 500, 1000, or 5000 nM.

    Article Snippet: Caspase inhibitor dissolved in DMSO (0, 0.1, 1, 10, 100, 1000, or 10000 nM) was combined with recombinant human caspase-3 enzyme (C1224-10UG, Sigma) (100 nM) in 1× phosphate buffered saline (PBS) in microcentrifuge tubes, vortexed, and incubated at 37°C for 30 min. After incubation, Caspase-Glo 3/7 reagent (Promega) was added in accordance with the manufacturer’s instructions.

    Techniques: In Vitro, Cell Cycle Assay, Flow Cytometry, Cytometry, Activity Assay, Western Blot

    [ 18 F]FB-VAD-FMK uptake reflects molecular response to Aurora B kinase inhibition in vivo Representative [ 18 F]FB-VAD-FMK transverse PET images of DLD-1/SW620 xenograft-bearing mice treated with vehicle or AZD-1152; tumors denoted by white arrows. Probe accumulation was absent in areas of central necrosis, as denoted by orange asterisks (A). Representative [ 18 F]FB-VAD-FMK time-activity curves for vehicle- and drug-treated DLD-1 and SW620 xenograft tumors. Vehicle- and drug-treated DLD-1 tumors exhibited similar washout, while greater retention in drug- versus vehicle-treated xenografts was observed for SW620 tumors (B). Quantification of tissue %ID/g revealed a statistical significant difference between vehicle- and drug-treated SW620 (p = 0.030) tumors but not for analogously treated DLD-1 tumors (p = 0.510) (C). Representative high-power white-light photo micrographs (40×) of caspase-3 immunohistochemical and H E stained DLD-1 and SW620 tissues obtained from xenografts collected immediately following imaging (D).

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: A Peptide-Based Positron Emission Tomography Probe for In Vivo Detection of Caspase Activity in Apoptotic Cells

    doi: 10.1158/1078-0432.CCR-13-2444

    Figure Lengend Snippet: [ 18 F]FB-VAD-FMK uptake reflects molecular response to Aurora B kinase inhibition in vivo Representative [ 18 F]FB-VAD-FMK transverse PET images of DLD-1/SW620 xenograft-bearing mice treated with vehicle or AZD-1152; tumors denoted by white arrows. Probe accumulation was absent in areas of central necrosis, as denoted by orange asterisks (A). Representative [ 18 F]FB-VAD-FMK time-activity curves for vehicle- and drug-treated DLD-1 and SW620 xenograft tumors. Vehicle- and drug-treated DLD-1 tumors exhibited similar washout, while greater retention in drug- versus vehicle-treated xenografts was observed for SW620 tumors (B). Quantification of tissue %ID/g revealed a statistical significant difference between vehicle- and drug-treated SW620 (p = 0.030) tumors but not for analogously treated DLD-1 tumors (p = 0.510) (C). Representative high-power white-light photo micrographs (40×) of caspase-3 immunohistochemical and H E stained DLD-1 and SW620 tissues obtained from xenografts collected immediately following imaging (D).

    Article Snippet: Caspase inhibitor dissolved in DMSO (0, 0.1, 1, 10, 100, 1000, or 10000 nM) was combined with recombinant human caspase-3 enzyme (C1224-10UG, Sigma) (100 nM) in 1× phosphate buffered saline (PBS) in microcentrifuge tubes, vortexed, and incubated at 37°C for 30 min. After incubation, Caspase-Glo 3/7 reagent (Promega) was added in accordance with the manufacturer’s instructions.

    Techniques: Inhibition, In Vivo, Positron Emission Tomography, Mouse Assay, Activity Assay, Immunohistochemistry, Staining, Imaging

    Effects of sulfhydration on cleavage of caspase‐3 or activity of cleaved caspase‐3. A, Activity of human recombinant caspase‐3 incubated with or without Na 2 S or STS with or without 2 mmol/L DTT at 37°C for 30 min. n=6 each; * or ** P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Thiosulfate Mediates Cytoprotective Effects of Hydrogen Sulfide Against Neuronal Ischemia

    doi: 10.1161/JAHA.115.002125

    Figure Lengend Snippet: Effects of sulfhydration on cleavage of caspase‐3 or activity of cleaved caspase‐3. A, Activity of human recombinant caspase‐3 incubated with or without Na 2 S or STS with or without 2 mmol/L DTT at 37°C for 30 min. n=6 each; * or ** P

    Article Snippet: Inhibition of purified recombinant human caspase‐3 activity by Na2 S or STS was examined by using the caspase‐3 inhibitor screening assay kit (EMD Millipore) according to the manufacturer's instructions with or without using (d,l )‐dithiothreitol (DTT) solution.

    Techniques: Activity Assay, Recombinant, Incubation

    H 2 S or STS inhibits mitochondrial pathway of apoptosis via modulating JNK pathway and Erk1/2 pathway in SH ‐ SY 5Y cells. Na 2 S at 0.5 mmol/L or STS at 0.25 mmol/L were added 5 h after the end of OGD . Protein expression levels were determined by immunoblotting with rabbit polyclonal antibody against (A) phosphorylated (Thr183/Tyr185) or total JNK , n=4 each, (B) phosphorylated (Thr202/Tyr204) or total Erk1/2, n=3 each, (C) phosphorylated (Ser112) or total Bad, n=3 each, (D) Bcl‐2, n=3 each, or vinculin, and (E) cleaved or total caspase‐3. n=7 each; *, **, or *** P

    Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

    Article Title: Thiosulfate Mediates Cytoprotective Effects of Hydrogen Sulfide Against Neuronal Ischemia

    doi: 10.1161/JAHA.115.002125

    Figure Lengend Snippet: H 2 S or STS inhibits mitochondrial pathway of apoptosis via modulating JNK pathway and Erk1/2 pathway in SH ‐ SY 5Y cells. Na 2 S at 0.5 mmol/L or STS at 0.25 mmol/L were added 5 h after the end of OGD . Protein expression levels were determined by immunoblotting with rabbit polyclonal antibody against (A) phosphorylated (Thr183/Tyr185) or total JNK , n=4 each, (B) phosphorylated (Thr202/Tyr204) or total Erk1/2, n=3 each, (C) phosphorylated (Ser112) or total Bad, n=3 each, (D) Bcl‐2, n=3 each, or vinculin, and (E) cleaved or total caspase‐3. n=7 each; *, **, or *** P

    Article Snippet: Inhibition of purified recombinant human caspase‐3 activity by Na2 S or STS was examined by using the caspase‐3 inhibitor screening assay kit (EMD Millipore) according to the manufacturer's instructions with or without using (d,l )‐dithiothreitol (DTT) solution.

    Techniques: Expressing

    KEA1–97 targets thioredoxin. (A) IsoTOP-ABPP analysis of KEA1–97 (10  μ M) in 231MFP breast cancer proteomes using DCT-alkyne probe. 231MFP breast cancer proteomes were pretreated with acetonitrile vehicle or KEA1–97 prior to labeling with DCT-alkyne (100  μ . (B) Gel-based ABPP studies showing competition of KEA1–97 against DCT-alkyne labeling (10  μ M) of pure human TXN. Shown is a representative gel and a dose−response curve. (C) TXN expression in GFP control or TXN-overexpressing 231MFP cells as assessed by qPCR. (D) Cell survival of 231MFP control or TXN-overexpressing cells treated with acetonitrile vehicle or KEA1–97 (10  μ M) for 24 h as assessed by Hoechst stain. (E) TXN activity assay. (F) Disruption of TXN interaction with caspase 3 upon treatment with KEA1–97 or mutation of TXN K72 to alanine. In the upper blot and left bar graph, pure His-tagged TXN and caspase 3 were preincubated with acetonitrile or KEA1–97 (100  μ M) prior to anti-His pulldown, SDS/PAGE, and blotting for caspase 3. In the lower blot and right bar graph, pure His-tagged TXN or TXN K72A mutant protein and caspase 3 were preincubated, subjected to anti-His pulldown, SDS/PAGE, and blotting for caspase 3. Data in B−E are shown as average ± SEM; data in A−F are  n  = 3/group. Significance is expressed as * p

    Journal: ACS chemical biology

    Article Title: Chemoproteomics-Enabled Covalent Ligand Screening Reveals a Thioredoxin-Caspase 3 Interaction Disruptor That Impairs Breast Cancer Pathogenicity

    doi: 10.1021/acschembio.7b00711

    Figure Lengend Snippet: KEA1–97 targets thioredoxin. (A) IsoTOP-ABPP analysis of KEA1–97 (10 μ M) in 231MFP breast cancer proteomes using DCT-alkyne probe. 231MFP breast cancer proteomes were pretreated with acetonitrile vehicle or KEA1–97 prior to labeling with DCT-alkyne (100 μ . (B) Gel-based ABPP studies showing competition of KEA1–97 against DCT-alkyne labeling (10 μ M) of pure human TXN. Shown is a representative gel and a dose−response curve. (C) TXN expression in GFP control or TXN-overexpressing 231MFP cells as assessed by qPCR. (D) Cell survival of 231MFP control or TXN-overexpressing cells treated with acetonitrile vehicle or KEA1–97 (10 μ M) for 24 h as assessed by Hoechst stain. (E) TXN activity assay. (F) Disruption of TXN interaction with caspase 3 upon treatment with KEA1–97 or mutation of TXN K72 to alanine. In the upper blot and left bar graph, pure His-tagged TXN and caspase 3 were preincubated with acetonitrile or KEA1–97 (100 μ M) prior to anti-His pulldown, SDS/PAGE, and blotting for caspase 3. In the lower blot and right bar graph, pure His-tagged TXN or TXN K72A mutant protein and caspase 3 were preincubated, subjected to anti-His pulldown, SDS/PAGE, and blotting for caspase 3. Data in B−E are shown as average ± SEM; data in A−F are n = 3/group. Significance is expressed as * p

    Article Snippet: Recombinant his-tagged TXN (Enzo Life Sciences Inc., ADI-SPP-892–200) was used as bait to precipitate pure recombinant caspase-3 (Origene Technologies Inc., TP304444) using Ni-NTA magnetic agarose beads (Qiagen, 220002−020).

    Techniques: Labeling, Expressing, Real-time Polymerase Chain Reaction, Staining, Activity Assay, Mutagenesis, SDS Page

    Apoptosis and antitumorigenic effects induced by KEA1–97 in 231MFP breast cancer cells. (A) Caspase 3/7 activation using a CellEvent Caspase 3/7 Green Detection Reagent. (B) KEA1–97 (10  μ M) induces apoptosis in 231MFP breast cancer cells assessed by propidium iodine and FITC Annexin-V staining and quantified by flow cytometry. (C) KEA1–97 treatment (5 mg/kg ip once per day) was initiated 16 days after the initiation of 231MFP tumor xenografts in immune-deficient SCID mice. Data in A−C are shown as average ± SEM. Data are  n  = 3/group in A and B, and  n  = 8 mice/group in C. Significance is expressed as * p

    Journal: ACS chemical biology

    Article Title: Chemoproteomics-Enabled Covalent Ligand Screening Reveals a Thioredoxin-Caspase 3 Interaction Disruptor That Impairs Breast Cancer Pathogenicity

    doi: 10.1021/acschembio.7b00711

    Figure Lengend Snippet: Apoptosis and antitumorigenic effects induced by KEA1–97 in 231MFP breast cancer cells. (A) Caspase 3/7 activation using a CellEvent Caspase 3/7 Green Detection Reagent. (B) KEA1–97 (10 μ M) induces apoptosis in 231MFP breast cancer cells assessed by propidium iodine and FITC Annexin-V staining and quantified by flow cytometry. (C) KEA1–97 treatment (5 mg/kg ip once per day) was initiated 16 days after the initiation of 231MFP tumor xenografts in immune-deficient SCID mice. Data in A−C are shown as average ± SEM. Data are n = 3/group in A and B, and n = 8 mice/group in C. Significance is expressed as * p

    Article Snippet: Recombinant his-tagged TXN (Enzo Life Sciences Inc., ADI-SPP-892–200) was used as bait to precipitate pure recombinant caspase-3 (Origene Technologies Inc., TP304444) using Ni-NTA magnetic agarose beads (Qiagen, 220002−020).

    Techniques: Activation Assay, Staining, Flow Cytometry, Cytometry, Mouse Assay

    Transactivation response DNA-binding protein 43 (TDP-43) fragments distinguish cell death pathways. ( A ) Primary cortical neurons were pretreated with calpain (30 μ mol/L SNJ1945, SJ) and caspase-3 (20 μ mol/L Z-VAD, ZD) inhibitors

    Journal: Journal of Cerebral Blood Flow & Metabolism

    Article Title: Dual vulnerability of TDP-43 to calpain and caspase-3 proteolysis after neurotoxic conditions and traumatic brain injury

    doi: 10.1038/jcbfm.2014.105

    Figure Lengend Snippet: Transactivation response DNA-binding protein 43 (TDP-43) fragments distinguish cell death pathways. ( A ) Primary cortical neurons were pretreated with calpain (30 μ mol/L SNJ1945, SJ) and caspase-3 (20 μ mol/L Z-VAD, ZD) inhibitors

    Article Snippet: In vitro digestion of cortex lysate (100 μ g) or purified recombinant human TDP-43 protein (3 μ g, OriGene, MD, USA) was performed with human erythrocyte calpain-1 (Calbiochem/EMD Bioscience, Billerica, MA, USA) and human recombinant caspase-3 (BD Pharmingen, San Jose, CA, USA), in a buffer containing 100 mmol/L Tris–HCl (pH 7.4), 20 mmol/L DTT, with or without 1 mmol/L CaCl2 , at room temperature for 30 minutes (calpain-1) or 4 hours (caspase-3).

    Techniques: Binding Assay

    Transactivation response DNA-binding protein 43 (TDP-43) in mouse or rat brain lysate is sensitive to in vitro calpain and caspase-3 digestion. The lysate of naïve mouse or rat cortex was in vitro cleaved by calpain-1 and caspase-3: Calpain-1

    Journal: Journal of Cerebral Blood Flow & Metabolism

    Article Title: Dual vulnerability of TDP-43 to calpain and caspase-3 proteolysis after neurotoxic conditions and traumatic brain injury

    doi: 10.1038/jcbfm.2014.105

    Figure Lengend Snippet: Transactivation response DNA-binding protein 43 (TDP-43) in mouse or rat brain lysate is sensitive to in vitro calpain and caspase-3 digestion. The lysate of naïve mouse or rat cortex was in vitro cleaved by calpain-1 and caspase-3: Calpain-1

    Article Snippet: In vitro digestion of cortex lysate (100 μ g) or purified recombinant human TDP-43 protein (3 μ g, OriGene, MD, USA) was performed with human erythrocyte calpain-1 (Calbiochem/EMD Bioscience, Billerica, MA, USA) and human recombinant caspase-3 (BD Pharmingen, San Jose, CA, USA), in a buffer containing 100 mmol/L Tris–HCl (pH 7.4), 20 mmol/L DTT, with or without 1 mmol/L CaCl2 , at room temperature for 30 minutes (calpain-1) or 4 hours (caspase-3).

    Techniques: Binding Assay, In Vitro

    Schematic of transactivation response DNA-binding protein 43 (TDP-43) proteolysis by calpain and caspase-3 pathways. In this model, caspase cleaves at the N-terminal (Asp89 ↓Ala90, Asp219 ↓Val220, based on Zhang YJ, et al ) producing caspase-dependent

    Journal: Journal of Cerebral Blood Flow & Metabolism

    Article Title: Dual vulnerability of TDP-43 to calpain and caspase-3 proteolysis after neurotoxic conditions and traumatic brain injury

    doi: 10.1038/jcbfm.2014.105

    Figure Lengend Snippet: Schematic of transactivation response DNA-binding protein 43 (TDP-43) proteolysis by calpain and caspase-3 pathways. In this model, caspase cleaves at the N-terminal (Asp89 ↓Ala90, Asp219 ↓Val220, based on Zhang YJ, et al ) producing caspase-dependent

    Article Snippet: In vitro digestion of cortex lysate (100 μ g) or purified recombinant human TDP-43 protein (3 μ g, OriGene, MD, USA) was performed with human erythrocyte calpain-1 (Calbiochem/EMD Bioscience, Billerica, MA, USA) and human recombinant caspase-3 (BD Pharmingen, San Jose, CA, USA), in a buffer containing 100 mmol/L Tris–HCl (pH 7.4), 20 mmol/L DTT, with or without 1 mmol/L CaCl2 , at room temperature for 30 minutes (calpain-1) or 4 hours (caspase-3).

    Techniques: Binding Assay

    Purified recombinant transactivation response DNA-binding protein 43 (TDP-43) fragmentation patterns by calpain or caspase-3. Recombinant human TDP-43 protein (3 μ g) was digested in vitro by calpain-1 or caspase-3. Intact protein and TDP-43

    Journal: Journal of Cerebral Blood Flow & Metabolism

    Article Title: Dual vulnerability of TDP-43 to calpain and caspase-3 proteolysis after neurotoxic conditions and traumatic brain injury

    doi: 10.1038/jcbfm.2014.105

    Figure Lengend Snippet: Purified recombinant transactivation response DNA-binding protein 43 (TDP-43) fragmentation patterns by calpain or caspase-3. Recombinant human TDP-43 protein (3 μ g) was digested in vitro by calpain-1 or caspase-3. Intact protein and TDP-43

    Article Snippet: In vitro digestion of cortex lysate (100 μ g) or purified recombinant human TDP-43 protein (3 μ g, OriGene, MD, USA) was performed with human erythrocyte calpain-1 (Calbiochem/EMD Bioscience, Billerica, MA, USA) and human recombinant caspase-3 (BD Pharmingen, San Jose, CA, USA), in a buffer containing 100 mmol/L Tris–HCl (pH 7.4), 20 mmol/L DTT, with or without 1 mmol/L CaCl2 , at room temperature for 30 minutes (calpain-1) or 4 hours (caspase-3).

    Techniques: Purification, Recombinant, Binding Assay, In Vitro

    Examination of relative contribution of calpains versus caspase-3 in tau proteolysis in rat cortex and hippocampus following TBI with tau-fragment-specific antibodies Western blot of naïve and CCI cortex ( A ) and hippocampus ( B ) time course samples similar to those of Figure 5 . However, these blots were probed with polyclonal antibody specific to caspase-mediated TauBDP-45K (lower panel), or polyclonal antibody specific to calpain-mediated TauBDP-35K (upper panel). Results shown are representative of three separate experiments.

    Journal: ASN NEURO

    Article Title: Dual vulnerability of tau to calpains and caspase-3 proteolysis under neurotoxic and neurodegenerative conditions

    doi: 10.1042/AN20100012

    Figure Lengend Snippet: Examination of relative contribution of calpains versus caspase-3 in tau proteolysis in rat cortex and hippocampus following TBI with tau-fragment-specific antibodies Western blot of naïve and CCI cortex ( A ) and hippocampus ( B ) time course samples similar to those of Figure 5 . However, these blots were probed with polyclonal antibody specific to caspase-mediated TauBDP-45K (lower panel), or polyclonal antibody specific to calpain-mediated TauBDP-35K (upper panel). Results shown are representative of three separate experiments.

    Article Snippet: In vitro protease digestion of naïve rat hippocampus lysate (30 μg) or purified recombinant human tau (Panvera Co., Madison, WI, U.S.A.) with purified proteases, human calpain-2 (BD Bioscience, Catalogue no. 208715, 1 μg/μl) and recombinant human caspase-3 (BD Bioscience, 1 unit/μl) was performed in a buffer containing 100 mM Tris/HCl (pH 7.4) and 20 mM DTT.

    Techniques: Western Blot

    Schematic of tau proteolysis by the calpain and caspase-3 pathways In this model, rat tau-4 repeat (4R) isoform with 432 residues is shown. It has four microtubule (MT)-binding repeats. Caspase-3 cleaves near the C-terminal (at Asp 412 ↓Ser 413 ), and an unknown N-terminal site producing key caspase fragment doublet of 45–48 kDa (TauBDP-45K). On the other hand, calpain cleaves at least three internal sites (Ser 120 ↓Lys 121 ; Pro 209 ↓Pro 2108 and Arg 380 ↓Glu 381 ) producing a key calpain fragment of 35 kDa (TauBDP-35K)

    Journal: ASN NEURO

    Article Title: Dual vulnerability of tau to calpains and caspase-3 proteolysis under neurotoxic and neurodegenerative conditions

    doi: 10.1042/AN20100012

    Figure Lengend Snippet: Schematic of tau proteolysis by the calpain and caspase-3 pathways In this model, rat tau-4 repeat (4R) isoform with 432 residues is shown. It has four microtubule (MT)-binding repeats. Caspase-3 cleaves near the C-terminal (at Asp 412 ↓Ser 413 ), and an unknown N-terminal site producing key caspase fragment doublet of 45–48 kDa (TauBDP-45K). On the other hand, calpain cleaves at least three internal sites (Ser 120 ↓Lys 121 ; Pro 209 ↓Pro 2108 and Arg 380 ↓Glu 381 ) producing a key calpain fragment of 35 kDa (TauBDP-35K)

    Article Snippet: In vitro protease digestion of naïve rat hippocampus lysate (30 μg) or purified recombinant human tau (Panvera Co., Madison, WI, U.S.A.) with purified proteases, human calpain-2 (BD Bioscience, Catalogue no. 208715, 1 μg/μl) and recombinant human caspase-3 (BD Bioscience, 1 unit/μl) was performed in a buffer containing 100 mM Tris/HCl (pH 7.4) and 20 mM DTT.

    Techniques: Binding Assay

    Tau protein in rat brain lysate is sensitive to in vitro calpain and caspase-3 digestion The lysate of naïve rat hippocampus was in vitro cleaved by calpain-1, calpain-2 and caspase-3: Control; Calpain-1 (1:500 protease/substrate ratio); calpain-2; (1:200 protease/substrate ratio); or caspase-3 digestion (1:50 protease/substrate ratio). The pattern of the tau protein fragmentation was monitored with total tau antibody ( A ) or antibodies specific to caspase-mediated TauBDP-45K ( B ) and calpain-mediated TauBDP-35K ( C ) respectively.

    Journal: ASN NEURO

    Article Title: Dual vulnerability of tau to calpains and caspase-3 proteolysis under neurotoxic and neurodegenerative conditions

    doi: 10.1042/AN20100012

    Figure Lengend Snippet: Tau protein in rat brain lysate is sensitive to in vitro calpain and caspase-3 digestion The lysate of naïve rat hippocampus was in vitro cleaved by calpain-1, calpain-2 and caspase-3: Control; Calpain-1 (1:500 protease/substrate ratio); calpain-2; (1:200 protease/substrate ratio); or caspase-3 digestion (1:50 protease/substrate ratio). The pattern of the tau protein fragmentation was monitored with total tau antibody ( A ) or antibodies specific to caspase-mediated TauBDP-45K ( B ) and calpain-mediated TauBDP-35K ( C ) respectively.

    Article Snippet: In vitro protease digestion of naïve rat hippocampus lysate (30 μg) or purified recombinant human tau (Panvera Co., Madison, WI, U.S.A.) with purified proteases, human calpain-2 (BD Bioscience, Catalogue no. 208715, 1 μg/μl) and recombinant human caspase-3 (BD Bioscience, 1 unit/μl) was performed in a buffer containing 100 mM Tris/HCl (pH 7.4) and 20 mM DTT.

    Techniques: In Vitro

    Intracellular protein delivery. Intracellular delivery of caspase 3 in MCF-7 cells after 48 h incubation at a fixed PTX concentration of 10 μg/mL and caspase-3 concentration of 76 nM. (A) WB assay: (1) saline, (2) CLG, (3) naked caspase 3, (4) Taxol, (5) PNPs, (6) PNPplex, (7) HA-PNPplex, (8) PUL, (9) PULplex. (B) Quantitative analysis ( n = 3, * P

    Journal: Theranostics

    Article Title: Drug-delivering-drug platform-mediated potent protein therapeutics via a non-endo-lysosomal route

    doi: 10.7150/thno.23804

    Figure Lengend Snippet: Intracellular protein delivery. Intracellular delivery of caspase 3 in MCF-7 cells after 48 h incubation at a fixed PTX concentration of 10 μg/mL and caspase-3 concentration of 76 nM. (A) WB assay: (1) saline, (2) CLG, (3) naked caspase 3, (4) Taxol, (5) PNPs, (6) PNPplex, (7) HA-PNPplex, (8) PUL, (9) PULplex. (B) Quantitative analysis ( n = 3, * P

    Article Snippet: Human recombinant caspase 3 was purchased from Cloud-Clone Corp. (USA).

    Techniques: Incubation, Concentration Assay, Western Blot

    Cell viability and apoptosis. Cytotoxicity of preparations against (A) MCF-7 without expression of the endogenous caspase 3 and (B) 4T1 with normal caspase 3 expression cells after 48 h of incubation with PTX at concentrations ranging from 0.5 to 100 µg/mL ( n = 5, * P

    Journal: Theranostics

    Article Title: Drug-delivering-drug platform-mediated potent protein therapeutics via a non-endo-lysosomal route

    doi: 10.7150/thno.23804

    Figure Lengend Snippet: Cell viability and apoptosis. Cytotoxicity of preparations against (A) MCF-7 without expression of the endogenous caspase 3 and (B) 4T1 with normal caspase 3 expression cells after 48 h of incubation with PTX at concentrations ranging from 0.5 to 100 µg/mL ( n = 5, * P

    Article Snippet: Human recombinant caspase 3 was purchased from Cloud-Clone Corp. (USA).

    Techniques: Expressing, Incubation

    Design and proposed active mechanism for the intracellular protein delivery platform based on rod-like pure drug nanoparticles (PNPs) with cellular entry bypassing endo-lysosomes. (1) Preparation of PNPs via antisolvent-precipitation. (2) Proteins, such as caspase 3 or BSA, are loaded on the PNPs surface to prepare PNPplex through electrostatic interaction. (3) Hyaluronic acid (HA) is used to further coat PNPplex and form HA-PNPplex, aiming to shield the positive charge and target CD44 receptors. The proposed procedure in vivo : (4) HA-PNPplex are administered via intravenous injection ( i.v. ), (5) accumulate in the tumor site, (6) enter cancer cells through CD44-receptor mediation via the caveosome pathway without entrapment in endo-lysosomes, and finally (7) release protein and drug in cytoplasm for disease therapy.

    Journal: Theranostics

    Article Title: Drug-delivering-drug platform-mediated potent protein therapeutics via a non-endo-lysosomal route

    doi: 10.7150/thno.23804

    Figure Lengend Snippet: Design and proposed active mechanism for the intracellular protein delivery platform based on rod-like pure drug nanoparticles (PNPs) with cellular entry bypassing endo-lysosomes. (1) Preparation of PNPs via antisolvent-precipitation. (2) Proteins, such as caspase 3 or BSA, are loaded on the PNPs surface to prepare PNPplex through electrostatic interaction. (3) Hyaluronic acid (HA) is used to further coat PNPplex and form HA-PNPplex, aiming to shield the positive charge and target CD44 receptors. The proposed procedure in vivo : (4) HA-PNPplex are administered via intravenous injection ( i.v. ), (5) accumulate in the tumor site, (6) enter cancer cells through CD44-receptor mediation via the caveosome pathway without entrapment in endo-lysosomes, and finally (7) release protein and drug in cytoplasm for disease therapy.

    Article Snippet: Human recombinant caspase 3 was purchased from Cloud-Clone Corp. (USA).

    Techniques: In Vivo, Injection

    Preparation and characterization. (A) Impact of PTX-loading ranging from 1 mg to 50 mg on the particle size of PNPs. (B) Native PAGE study of caspase 3 or RITC-BSA loaded in PNPplex. The mass ratio of CLG and caspase 3 or RITC-BSA was increased from 1 to 64. (C) Particle size and PDI of the nanoparticles loading caspase 3 or BSA. (D) TEM image of HA-PNPplex. (E) Fluorescence emission spectra for FRET assay. FITC and RITC were used as the donor and acceptor, respectively. Dual-labeled HA-PNPplex (FITC-RITC-HA-PNPplex) were prepared by assembling FITC-BSA on RITC-PNPs followed by HA coating. Excitation wavelength: 492 nm. FRET effect is demonstrated via the reduction fluorescence intensity of donor at 520 nm with increased fluorescence of acceptor at 590 nm. CD spectra of functional proteins: (F-G) encapsulated proteins and (H) released caspase 3. The control was naked functional protein.

    Journal: Theranostics

    Article Title: Drug-delivering-drug platform-mediated potent protein therapeutics via a non-endo-lysosomal route

    doi: 10.7150/thno.23804

    Figure Lengend Snippet: Preparation and characterization. (A) Impact of PTX-loading ranging from 1 mg to 50 mg on the particle size of PNPs. (B) Native PAGE study of caspase 3 or RITC-BSA loaded in PNPplex. The mass ratio of CLG and caspase 3 or RITC-BSA was increased from 1 to 64. (C) Particle size and PDI of the nanoparticles loading caspase 3 or BSA. (D) TEM image of HA-PNPplex. (E) Fluorescence emission spectra for FRET assay. FITC and RITC were used as the donor and acceptor, respectively. Dual-labeled HA-PNPplex (FITC-RITC-HA-PNPplex) were prepared by assembling FITC-BSA on RITC-PNPs followed by HA coating. Excitation wavelength: 492 nm. FRET effect is demonstrated via the reduction fluorescence intensity of donor at 520 nm with increased fluorescence of acceptor at 590 nm. CD spectra of functional proteins: (F-G) encapsulated proteins and (H) released caspase 3. The control was naked functional protein.

    Article Snippet: Human recombinant caspase 3 was purchased from Cloud-Clone Corp. (USA).

    Techniques: Clear Native PAGE, Transmission Electron Microscopy, Fluorescence, Labeling, Functional Assay

    Relative activity of caspase-8 with model peptides containing serine or phosphoserine at each position from P4 to P3′. Peptides modeled after the caspase-8 cleavage site of pro-caspase-3 were incubated with caspase-8 (100 n m ) for 5 min at 37 °C before the reaction was terminated by an excess of the irreversible caspase inhibitor zVAD-fmk. Error bars represent the standard deviation of four reactions. Means of the caspase activities toward the phosphorylated and the corresponding nonphosphorylated peptide were compared using analysis of variance and Tukey's test. All pairs had p values

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation *

    doi: 10.1074/mcp.M113.037374

    Figure Lengend Snippet: Relative activity of caspase-8 with model peptides containing serine or phosphoserine at each position from P4 to P3′. Peptides modeled after the caspase-8 cleavage site of pro-caspase-3 were incubated with caspase-8 (100 n m ) for 5 min at 37 °C before the reaction was terminated by an excess of the irreversible caspase inhibitor zVAD-fmk. Error bars represent the standard deviation of four reactions. Means of the caspase activities toward the phosphorylated and the corresponding nonphosphorylated peptide were compared using analysis of variance and Tukey's test. All pairs had p values

    Article Snippet: Caspase Assays Recombinant caspase-3 (Addgene plasmid number 11821 ( )), caspase-7 (Addgene plasmid number 11825 ( )), and caspase-8 (Addgene plasmid number 11827 ( )) were purified and their activity was assessed via active site titration as described previously ( ).

    Techniques: Activity Assay, Incubation, Standard Deviation

    Relative activities of caspase-3 and caspase-7 with model and phosphopeptides containing serine residues positioned within the P5–P4′ consensus motif for caspase cleavage. Caspase cleavage of peptide substrates was measured using fluorescence of an internally quenched tryptophan residue at 355 nm after excitation at 280 nm. Peptides or phosphopeptides modeled after Golgin-160 ( A ) and PARP1 ( B ) were assayed with caspase-3 or caspase-7 for 10 min at 37 °C and stopped by an excess of the irreversible caspase inhibitor zVAD-fmk. Error bars represent the standard deviation of four reactions. A , means of the caspase activities toward phosphorylated peptides and their nonphosphorylated counterparts were compared using analysis of variance and Tukey's test. For caspase-3, P4, P3, P1′, P2′, P3′, and P4′ phosphopeptide versus nonphosphorylated peptide pairs had p values

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation *

    doi: 10.1074/mcp.M113.037374

    Figure Lengend Snippet: Relative activities of caspase-3 and caspase-7 with model and phosphopeptides containing serine residues positioned within the P5–P4′ consensus motif for caspase cleavage. Caspase cleavage of peptide substrates was measured using fluorescence of an internally quenched tryptophan residue at 355 nm after excitation at 280 nm. Peptides or phosphopeptides modeled after Golgin-160 ( A ) and PARP1 ( B ) were assayed with caspase-3 or caspase-7 for 10 min at 37 °C and stopped by an excess of the irreversible caspase inhibitor zVAD-fmk. Error bars represent the standard deviation of four reactions. A , means of the caspase activities toward phosphorylated peptides and their nonphosphorylated counterparts were compared using analysis of variance and Tukey's test. For caspase-3, P4, P3, P1′, P2′, P3′, and P4′ phosphopeptide versus nonphosphorylated peptide pairs had p values

    Article Snippet: Caspase Assays Recombinant caspase-3 (Addgene plasmid number 11821 ( )), caspase-7 (Addgene plasmid number 11825 ( )), and caspase-8 (Addgene plasmid number 11827 ( )) were purified and their activity was assessed via active site titration as described previously ( ).

    Techniques: Fluorescence, Standard Deviation

    TAILS analysis revealed MST3, Golgin-160, and Yap1 as validated candidates in which cleavage is regulated by phosphorylation. A , caspase recognition motif in MST3, Yap1, and Golgin-160. MS/MS analysis of MST3 ( B ), Yap1 ( C ), and Golgin-160 ( D ). E , HeLa lysates were treated identically to those samples prepared for N-terminomic analysis (as described in “Experimental Procedures”), except when they were utilized for Western blotting with antibodies directed against MST3, Yap1, or Golgin-160 as indicated. Caspase-3 and caspase-7 were used at concentrations of 0, 50, 500, and 5000 n m . The asterisk denotes a nonspecific band, and the arrow and arrowhead bands were differentially regulated by lysate dephosphorylation. F , HeLa cells were treated for 3 h with 1 μ m staurosporine and/or 500 n m okadaic acid prior to lysis. Lysates were analyzed using immunoblots with the indicated antibodies. PARP1 cleavage was used as a control for caspase activation. The asterisk denotes a nonspecific band.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation *

    doi: 10.1074/mcp.M113.037374

    Figure Lengend Snippet: TAILS analysis revealed MST3, Golgin-160, and Yap1 as validated candidates in which cleavage is regulated by phosphorylation. A , caspase recognition motif in MST3, Yap1, and Golgin-160. MS/MS analysis of MST3 ( B ), Yap1 ( C ), and Golgin-160 ( D ). E , HeLa lysates were treated identically to those samples prepared for N-terminomic analysis (as described in “Experimental Procedures”), except when they were utilized for Western blotting with antibodies directed against MST3, Yap1, or Golgin-160 as indicated. Caspase-3 and caspase-7 were used at concentrations of 0, 50, 500, and 5000 n m . The asterisk denotes a nonspecific band, and the arrow and arrowhead bands were differentially regulated by lysate dephosphorylation. F , HeLa cells were treated for 3 h with 1 μ m staurosporine and/or 500 n m okadaic acid prior to lysis. Lysates were analyzed using immunoblots with the indicated antibodies. PARP1 cleavage was used as a control for caspase activation. The asterisk denotes a nonspecific band.

    Article Snippet: Caspase Assays Recombinant caspase-3 (Addgene plasmid number 11821 ( )), caspase-7 (Addgene plasmid number 11825 ( )), and caspase-8 (Addgene plasmid number 11827 ( )) were purified and their activity was assessed via active site titration as described previously ( ).

    Techniques: Mass Spectrometry, Western Blot, De-Phosphorylation Assay, Lysis, Activation Assay

    Caspase activities toward synthetic peptides modeled after substrates identified via TAILS. Synthetic peptides or phosphopeptides modeled after Yap1 ( A ), Golgin-160 ( B ), or MST3 ( C ) were treated with or without λ phosphatase as indicated prior to incubation with caspase-3 or caspase-7. Fluorescence of an internally quenched tryptophan residue at 355 nm after excitation at 280 nm was measured to assess caspase cleavage of peptide substrates. Error bars represent the standard deviation of four reactions. Means for the caspase activities with phosphorylated peptides and their unphosphorylated counterparts were compared using analysis of variance and Tukey's test. Control versus phosphatase-treated pairs for phosphopeptides all had p values

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation *

    doi: 10.1074/mcp.M113.037374

    Figure Lengend Snippet: Caspase activities toward synthetic peptides modeled after substrates identified via TAILS. Synthetic peptides or phosphopeptides modeled after Yap1 ( A ), Golgin-160 ( B ), or MST3 ( C ) were treated with or without λ phosphatase as indicated prior to incubation with caspase-3 or caspase-7. Fluorescence of an internally quenched tryptophan residue at 355 nm after excitation at 280 nm was measured to assess caspase cleavage of peptide substrates. Error bars represent the standard deviation of four reactions. Means for the caspase activities with phosphorylated peptides and their unphosphorylated counterparts were compared using analysis of variance and Tukey's test. Control versus phosphatase-treated pairs for phosphopeptides all had p values

    Article Snippet: Caspase Assays Recombinant caspase-3 (Addgene plasmid number 11821 ( )), caspase-7 (Addgene plasmid number 11825 ( )), and caspase-8 (Addgene plasmid number 11827 ( )) were purified and their activity was assessed via active site titration as described previously ( ).

    Techniques: Incubation, Fluorescence, Standard Deviation

    Cisplatin-induced p70S6K downregulation is mediated by caspase-3. (a), A549 cells were treated with 10 µM Mg132, 20 µM calpeptin and 20 µM z-VAD for 30 min prior to incubation with 30 μM cisplatin. (b), A549 cells were treated with 20 μM caspase inhibitors for 30 min prior to incubation with 30 μM cisplatin. z-VAD, broad specificity caspase inhibitor; z-VDVAD, caspase-2 inhibitor; z-DEVD, caspase-3 inhibitor; z-IETD, caspase-8 inhibitor; z-LEHD, caspase-9 inhibitor. Cells were processed following treatment with cisplatin for 24 h. Western blot analysis was performed with total cellular extracts using indicated antibodies. GAPDH was used as a loading control. The arrow indicates cleaved p70S6K band. Results are representative of two independent experiments. (c), A549 cells were transfected with control non-targeting siRNA or siRNA SMARTpool against caspase-2 or caspase-3 as described in “Experimental Procedures.” Western blot analysis was performed with total cellular extracts using indicated antibodies. (d), A549 cells transfected with control and caspase-2 siRNA were treated with or without cisplatin for 24 h and Western blot analysis was performed with total cell extracts. GAPDH was used as a loading control. The arrow indicates cleaved p70S6K band. Results are indicative of two independent experiments.

    Journal: Biochemistry

    Article Title: Proteolytic cleavage of p70 ribosomal S6 kinase by caspase-3 during DNA damage-induced apoptosis

    doi: 10.1021/bi801840s

    Figure Lengend Snippet: Cisplatin-induced p70S6K downregulation is mediated by caspase-3. (a), A549 cells were treated with 10 µM Mg132, 20 µM calpeptin and 20 µM z-VAD for 30 min prior to incubation with 30 μM cisplatin. (b), A549 cells were treated with 20 μM caspase inhibitors for 30 min prior to incubation with 30 μM cisplatin. z-VAD, broad specificity caspase inhibitor; z-VDVAD, caspase-2 inhibitor; z-DEVD, caspase-3 inhibitor; z-IETD, caspase-8 inhibitor; z-LEHD, caspase-9 inhibitor. Cells were processed following treatment with cisplatin for 24 h. Western blot analysis was performed with total cellular extracts using indicated antibodies. GAPDH was used as a loading control. The arrow indicates cleaved p70S6K band. Results are representative of two independent experiments. (c), A549 cells were transfected with control non-targeting siRNA or siRNA SMARTpool against caspase-2 or caspase-3 as described in “Experimental Procedures.” Western blot analysis was performed with total cellular extracts using indicated antibodies. (d), A549 cells transfected with control and caspase-2 siRNA were treated with or without cisplatin for 24 h and Western blot analysis was performed with total cell extracts. GAPDH was used as a loading control. The arrow indicates cleaved p70S6K band. Results are indicative of two independent experiments.

    Article Snippet: Active human recombinant caspase-3 was obtained from Axora (San Diego, CA).

    Techniques: Incubation, Western Blot, Transfection

    In vitro-translated p70S6K is cleaved by caspase-3. EE-tagged p70S6K cloned into pcDNA3 was synthesized with the T7 Quick TNT kit (Promega) in the presence of [35S]Met and incubated with or without human recombinant caspase-3 as described in the “Experimental Procedures.” The reaction products were separated by SDS-PAGE and analyzed by autoradiography. Arrows indicate cleaved p70S6K bands. The results are representative of three separate experiments.

    Journal: Biochemistry

    Article Title: Proteolytic cleavage of p70 ribosomal S6 kinase by caspase-3 during DNA damage-induced apoptosis

    doi: 10.1021/bi801840s

    Figure Lengend Snippet: In vitro-translated p70S6K is cleaved by caspase-3. EE-tagged p70S6K cloned into pcDNA3 was synthesized with the T7 Quick TNT kit (Promega) in the presence of [35S]Met and incubated with or without human recombinant caspase-3 as described in the “Experimental Procedures.” The reaction products were separated by SDS-PAGE and analyzed by autoradiography. Arrows indicate cleaved p70S6K bands. The results are representative of three separate experiments.

    Article Snippet: Active human recombinant caspase-3 was obtained from Axora (San Diego, CA).

    Techniques: In Vitro, Clone Assay, Synthesized, Incubation, Recombinant, SDS Page, Autoradiography

    Cisplatin fails to cleave p70S6K in MCF-7 cells. (a), MCF-7 cells were treated with indicated concentrations of cisplatin. (b), MCF-7 cells were transfected with pcDNA3 vector alone or vector containing caspase-3 construct and then treated with 25 μM cisplatin for 24 h. Cells were then processed and Western blot analysis was performed with total cellular extracts using indicated antibodies. GAPDH was used as a loading control. The arrow indicates cleaved p70S6K band.

    Journal: Biochemistry

    Article Title: Proteolytic cleavage of p70 ribosomal S6 kinase by caspase-3 during DNA damage-induced apoptosis

    doi: 10.1021/bi801840s

    Figure Lengend Snippet: Cisplatin fails to cleave p70S6K in MCF-7 cells. (a), MCF-7 cells were treated with indicated concentrations of cisplatin. (b), MCF-7 cells were transfected with pcDNA3 vector alone or vector containing caspase-3 construct and then treated with 25 μM cisplatin for 24 h. Cells were then processed and Western blot analysis was performed with total cellular extracts using indicated antibodies. GAPDH was used as a loading control. The arrow indicates cleaved p70S6K band.

    Article Snippet: Active human recombinant caspase-3 was obtained from Axora (San Diego, CA).

    Techniques: Transfection, Plasmid Preparation, Construct, Western Blot

    The proteolytic cleavage of p70S6K by caspase-3 enhanced cisplatin-induced cell death. (a), A representative cartoon showing the p70S6K mutants that were generated. (b), HeLa cells were transfected with an empty vector or EE-tagged WT or mutant p70S6K as described under “Experimental Procedures.” Cells were treated with 2 μM cisplatin for 18 h and Western blot analysis was performed with antibody against EE epitope. GAPDH was used to control for loading differences. (c), Nuclei were stained with propidium iodide and analyzed using a flow cytometer. Results are representative of three separate experiments.

    Journal: Biochemistry

    Article Title: Proteolytic cleavage of p70 ribosomal S6 kinase by caspase-3 during DNA damage-induced apoptosis

    doi: 10.1021/bi801840s

    Figure Lengend Snippet: The proteolytic cleavage of p70S6K by caspase-3 enhanced cisplatin-induced cell death. (a), A representative cartoon showing the p70S6K mutants that were generated. (b), HeLa cells were transfected with an empty vector or EE-tagged WT or mutant p70S6K as described under “Experimental Procedures.” Cells were treated with 2 μM cisplatin for 18 h and Western blot analysis was performed with antibody against EE epitope. GAPDH was used to control for loading differences. (c), Nuclei were stained with propidium iodide and analyzed using a flow cytometer. Results are representative of three separate experiments.

    Article Snippet: Active human recombinant caspase-3 was obtained from Axora (San Diego, CA).

    Techniques: Generated, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Staining, Flow Cytometry, Cytometry

    TDP-43 D169G mutant is more susceptible to proteolytic cleavage by caspase 3. ( A ) The wild-type, A90V and D169G mutant of TDP-43 (N-RRM12, residues 1–265) were incubated with caspase 3 for two hours. The SDS-PAGE stained with Coomassie blue revealed that TDP-43 was cleaved into TDP-35. Tandem mass spectrometry confirmed that caspase 3 digested N-RRM12 into two major fragments, TDP-35 (residues 90–265), and residues 1–89. ( B ) The wild-type, A90V and D169G mutant of TDP-43 (N-RRM12) were incubated with caspase 4, 6 and 7 for 16 hours. The digested proteins were resolved by 15% Tricine-SDS-PAGE with Coomassie blue staining. The mean percentages with standard errors shown at the bottom of the gel were calculated from three independent experiments. Statistical significance P values were determined by an unpaired two-tailed Student’s t -test.

    Journal: Scientific Reports

    Article Title: Structural analysis of disease-related TDP-43 D169G mutation: linking enhanced stability and caspase cleavage efficiency to protein accumulation

    doi: 10.1038/srep21581

    Figure Lengend Snippet: TDP-43 D169G mutant is more susceptible to proteolytic cleavage by caspase 3. ( A ) The wild-type, A90V and D169G mutant of TDP-43 (N-RRM12, residues 1–265) were incubated with caspase 3 for two hours. The SDS-PAGE stained with Coomassie blue revealed that TDP-43 was cleaved into TDP-35. Tandem mass spectrometry confirmed that caspase 3 digested N-RRM12 into two major fragments, TDP-35 (residues 90–265), and residues 1–89. ( B ) The wild-type, A90V and D169G mutant of TDP-43 (N-RRM12) were incubated with caspase 4, 6 and 7 for 16 hours. The digested proteins were resolved by 15% Tricine-SDS-PAGE with Coomassie blue staining. The mean percentages with standard errors shown at the bottom of the gel were calculated from three independent experiments. Statistical significance P values were determined by an unpaired two-tailed Student’s t -test.

    Article Snippet: In vitro caspase digestion assays The TDP-43 (N-RRM12) (5 μg) and its mutants were incubated respectively with human active recombinant caspase 3 (BD Phamingen), caspase 4 (BioVision), caspase 6 (BioVision), and caspase 7 (BioVision) in a buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 10% glycerol and 0.1% CHAPS at 37 °C for 2 hours (caspase 4, 6, 7 for 16 hours).

    Techniques: Mutagenesis, Incubation, SDS Page, Staining, Mass Spectrometry, Two Tailed Test

    Protein expression of cleaved caspase-3. Western blots for cleaved caspase-3 in naïve, sham, hypoxia–ischemia injured+hypothermia (HI+HypoT), HI+rapid rewarming (HI+rapid RW), and HI+slow rewarming

    Journal: Journal of Cerebral Blood Flow & Metabolism

    Article Title: Rewarming from therapeutic hypothermia induces cortical neuron apoptosis in a swine model of neonatal hypoxic–ischemic encephalopathy

    doi: 10.1038/jcbfm.2014.245

    Figure Lengend Snippet: Protein expression of cleaved caspase-3. Western blots for cleaved caspase-3 in naïve, sham, hypoxia–ischemia injured+hypothermia (HI+HypoT), HI+rapid rewarming (HI+rapid RW), and HI+slow rewarming

    Article Snippet: We tested multiple caspase-3 antibodies in piglet tissue by western blotting and compared the bands with a recombinant caspase-3 positive control (Biovision).

    Techniques: Expressing, Western Blot

    The piglets underwent hypoxia–ischemia followed by hypothermia and rapid rewarming. In comparison to artificial CSF (aCSF), subdural administration of caspase-3 inhibitor decreased the number of ( A ) apoptotic profiles by hematoxylin and eosin

    Journal: Journal of Cerebral Blood Flow & Metabolism

    Article Title: Rewarming from therapeutic hypothermia induces cortical neuron apoptosis in a swine model of neonatal hypoxic–ischemic encephalopathy

    doi: 10.1038/jcbfm.2014.245

    Figure Lengend Snippet: The piglets underwent hypoxia–ischemia followed by hypothermia and rapid rewarming. In comparison to artificial CSF (aCSF), subdural administration of caspase-3 inhibitor decreased the number of ( A ) apoptotic profiles by hematoxylin and eosin

    Article Snippet: We tested multiple caspase-3 antibodies in piglet tissue by western blotting and compared the bands with a recombinant caspase-3 positive control (Biovision).

    Techniques:

    Purified recombinant CK2 phosphorylates murine caspase-9 at Ser 348 in vitro . A , amino acid sequence in murine caspase-9 between the active site, QACGG ( * ), and the caspase-3 processing site. Arrows show the auto-processing and primary caspase-8 cleavage site, SEPD, and the caspase-3 site, DQLD. The filled black circle shows serine 348 predicted to be the target residue within a strong CK2 consensus motif ( underlined ). B–D, in vitro translated mC-9 protein was immunoprecipitated with anti-caspase-9 antibody and incubated in kinase buffer in the presence or absence of purified CK2. B , phosphorylation of mC-9 in the presence of CK2 inhibitors ( lanes 3–5 ): DRB, heparin, and apigenin. C, top panel , CK2 phosphorylation of mC-9 and the LDAD mutant in the presence or absence of DRB in vitro. Lower panel , immunoprecipitation ( IP )/Western of cold in vitro translated mC-9 and LDAD. D , CK2 phosphorylation of mC-9 and mutants AEPD and LDVD in vitro .

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation of Murine Caspase-9 by the Protein Kinase Casein Kinase 2 Regulates Its Cleavage by Caspase-8 *Phosphorylation of Murine Caspase-9 by the Protein Kinase Casein Kinase 2 Regulates Its Cleavage by Caspase-8 * S⃞

    doi: 10.1074/jbc.M802846200

    Figure Lengend Snippet: Purified recombinant CK2 phosphorylates murine caspase-9 at Ser 348 in vitro . A , amino acid sequence in murine caspase-9 between the active site, QACGG ( * ), and the caspase-3 processing site. Arrows show the auto-processing and primary caspase-8 cleavage site, SEPD, and the caspase-3 site, DQLD. The filled black circle shows serine 348 predicted to be the target residue within a strong CK2 consensus motif ( underlined ). B–D, in vitro translated mC-9 protein was immunoprecipitated with anti-caspase-9 antibody and incubated in kinase buffer in the presence or absence of purified CK2. B , phosphorylation of mC-9 in the presence of CK2 inhibitors ( lanes 3–5 ): DRB, heparin, and apigenin. C, top panel , CK2 phosphorylation of mC-9 and the LDAD mutant in the presence or absence of DRB in vitro. Lower panel , immunoprecipitation ( IP )/Western of cold in vitro translated mC-9 and LDAD. D , CK2 phosphorylation of mC-9 and mutants AEPD and LDVD in vitro .

    Article Snippet: Active recombinant caspase-3 and caspase-8 were purchased from Pharmingen.

    Techniques: Purification, Recombinant, In Vitro, Sequencing, Immunoprecipitation, Incubation, Mutagenesis, Western Blot

    Phosphorylation of PS-2 inhibits its cleavage by caspase-3. ( a ) HeLa cells stably expressing PS-2 CTF wt were labeled with [ 32 P]orthophosphate, and the PS-2 CTFs were immunoprecipitated with the anti-Flag antibody. The immunoprecipitate was divided into two aliquots and incubated in the presence or absence of purified caspase-3. Proteins were separated by SDS/PAGE and transferred to a poly(vinylidene difluoride) membrane. Radiolabeled proteins were visualized by PhosphorImaging ( Left ). Subsequently, the same membrane was probed with the anti-Flag antibody ( Right ). Note that caspase-3 cleaves selectively the unphosphorylated form of PS-2 CTF. ( b ) Mimicking phosphorylation by substitution of serines 327 and 330 by aspartate residues also inhibits cleavage by caspase-3. Fusion proteins of MBP with the respective PS-2 Loop domain (wt, S327/330A, or S327/330D) were incubated in the presence or absence of caspase-3. Full-length fusion proteins (PS-2-MBP) and cleavage products (PS-2-MBP Casp ) were detected by immunoblotting with antibody 3711. Fusion protein carrying the PS-2 Loop wt or the serine-to-alanine mutations were cleaved by caspase-3. In contrast, the introduction of negatively charged aspartate residues, which mimic phosphorylated serines, inhibits caspase-mediated cleavage. MBP itself was not cleaved by caspase-3 (data not shown). Similar results were obtained with purified active caspase-7 (although caspase-7 cleaves PS-2 with less efficiency than caspase-3; data not shown).

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

    Article Title: Phosphorylation of presenilin-2 regulates its cleavage by caspases and retards progression of apoptosis

    doi:

    Figure Lengend Snippet: Phosphorylation of PS-2 inhibits its cleavage by caspase-3. ( a ) HeLa cells stably expressing PS-2 CTF wt were labeled with [ 32 P]orthophosphate, and the PS-2 CTFs were immunoprecipitated with the anti-Flag antibody. The immunoprecipitate was divided into two aliquots and incubated in the presence or absence of purified caspase-3. Proteins were separated by SDS/PAGE and transferred to a poly(vinylidene difluoride) membrane. Radiolabeled proteins were visualized by PhosphorImaging ( Left ). Subsequently, the same membrane was probed with the anti-Flag antibody ( Right ). Note that caspase-3 cleaves selectively the unphosphorylated form of PS-2 CTF. ( b ) Mimicking phosphorylation by substitution of serines 327 and 330 by aspartate residues also inhibits cleavage by caspase-3. Fusion proteins of MBP with the respective PS-2 Loop domain (wt, S327/330A, or S327/330D) were incubated in the presence or absence of caspase-3. Full-length fusion proteins (PS-2-MBP) and cleavage products (PS-2-MBP Casp ) were detected by immunoblotting with antibody 3711. Fusion protein carrying the PS-2 Loop wt or the serine-to-alanine mutations were cleaved by caspase-3. In contrast, the introduction of negatively charged aspartate residues, which mimic phosphorylated serines, inhibits caspase-mediated cleavage. MBP itself was not cleaved by caspase-3 (data not shown). Similar results were obtained with purified active caspase-7 (although caspase-7 cleaves PS-2 with less efficiency than caspase-3; data not shown).

    Article Snippet: PS-2 CTFs, immunoprecipitated from cell lysates, or 1 μg of the respective fusionprotein PS-2Loop -MBP were incubated for 4 h at 37°C in 25 μl of caspase assay buffer (20 mM Hepes/100 mM sodium chloride/10 mM DTT/10 mM magnesium chloride/1 mM EDTA/0.1% CHAPS/10% sucrose, pH 7.2) in the presence or absence of 20 ng of recombinant active caspase-3 (PharMingen).

    Techniques: Stable Transfection, Expressing, Labeling, Immunoprecipitation, Incubation, Purification, SDS Page

    (A) Immunoblot for endogenous pro-caspase-3 in hypoxic retinal cells treated without ( lane 2 ) and with enzyme inhibitors ( lanes 3–6 ). Intact pro-caspase-3 is indicated at 33 kDa ( filled arrowhead ) along with calpain-dependent breakdown products

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Calpain, Not Caspase, Is the Causative Protease for Hypoxic Damage in Cultured Monkey Retinal Cells

    doi: 10.1167/iovs.11-7497

    Figure Lengend Snippet: (A) Immunoblot for endogenous pro-caspase-3 in hypoxic retinal cells treated without ( lane 2 ) and with enzyme inhibitors ( lanes 3–6 ). Intact pro-caspase-3 is indicated at 33 kDa ( filled arrowhead ) along with calpain-dependent breakdown products

    Article Snippet: Active human recombinant caspase-3 (BioVision, Inc., Mountain View, CA) was used as a reference for the migration positions of the 12- and 17-kDa bands for activated caspase-3.

    Techniques:

    Orthohantaviruses N protein contains cleavage sites for caspase-3 and weakly inhibits caspase-3 activity. ( a ) Western blot analyses showing two cleaved fragments of the different orthohantavirus N protein after incubation with or without recombinant caspase-3 in the presence or absence of the caspase-3-inhibitor Ac-DEVD-CHO. The monoclonal antibody 7A2/D5 was used in order to detect the cleaved fragments of N protein. One representative experiment out of three is shown. ( b ) Caspase-3 activity after incubation with recombinant ANDV, PUUV or DOBV N protein (rANDV-N, rPUUV-N or rDOBV-N), or with recombinant control protein (DHFR). Data shown represent mean ± SD of three independent experiments. Paired t-test: ns non-significant; *p

    Journal: Scientific Reports

    Article Title: Orthohantaviruses belonging to three phylogroups all inhibit apoptosis in infected target cells

    doi: 10.1038/s41598-018-37446-1

    Figure Lengend Snippet: Orthohantaviruses N protein contains cleavage sites for caspase-3 and weakly inhibits caspase-3 activity. ( a ) Western blot analyses showing two cleaved fragments of the different orthohantavirus N protein after incubation with or without recombinant caspase-3 in the presence or absence of the caspase-3-inhibitor Ac-DEVD-CHO. The monoclonal antibody 7A2/D5 was used in order to detect the cleaved fragments of N protein. One representative experiment out of three is shown. ( b ) Caspase-3 activity after incubation with recombinant ANDV, PUUV or DOBV N protein (rANDV-N, rPUUV-N or rDOBV-N), or with recombinant control protein (DHFR). Data shown represent mean ± SD of three independent experiments. Paired t-test: ns non-significant; *p

    Article Snippet: Virus nucleocapsid protein cleavage assay Viruses, heat-inactivated at 96 °C for 10 minutes, were incubated with active human recombinant caspase-3 (with or without caspase-3 inhibitor) or active human recombinant granzyme B, in reaction buffer (BioVision) at 37 °C for 1 hour or as stated in the text.

    Techniques: Activity Assay, Western Blot, Incubation, Recombinant

    Orthohantaviruses hinder staurosporine-induced apoptosis in human primary endothelial cells. ( a ) Representative immunofluorescence images from three independent experiments of ANDV-, HTNV-, SEOV-, PUUV-, PHV- and TULV-infected HUVEC after exposure to staurosporine (2 μM) for approximately 4 hours. Cells were infected at a low MOI (0.01) to achieve 20 to 30% infection at 4 days post-infection. Following staurosporine-treatment, cells were stained with TUNEL (red) to assess apoptosis, convalescent PUUV patient serum (green) to detect virus infection and DAPI (blue) for nuclear counterstaining. Scale bar, 20 μm. ( b ) Graphs showing TUNEL-positive uninfected and infected cells. Data shown represent the mean ± SD of three independent experiments. ( c ) Western blot analyses of PARP (full-length and cleaved) in total cell lysate from infected and uninfected cells exposed to staurosporine (2 μM) for approximately 4 hours or until observing the effects of staurosporine in treated control cells. MOI 1 of virus was used, resulting in ≥95% of the cells being infected 4 days after infection (Supplementary Fig. 1 ). Specific monoclonal antibodies (mabs 1C12 for ANDV, HTNV, SEOV, PUUV and TULV, and 7B3F7 for PHV) were used for detection of N protein. Calnexin was used as loading control. One representative experiment out of three independent experiments is shown. Band intensity was analysed by densitometry of caspase-3 cleaved PARP and full-length PARP. The ratios in intensity between cleaved and full-length PARP were calculated and compared between infected and uninfected conditions. The ratio from uninfected cells represents maximal caspase-3 cleaved PARP/full-length PARP ratio (100%). The graphs show mean ± SD of the ratio between cleaved and full-length PARP from three experiments. Paired t-test: *p

    Journal: Scientific Reports

    Article Title: Orthohantaviruses belonging to three phylogroups all inhibit apoptosis in infected target cells

    doi: 10.1038/s41598-018-37446-1

    Figure Lengend Snippet: Orthohantaviruses hinder staurosporine-induced apoptosis in human primary endothelial cells. ( a ) Representative immunofluorescence images from three independent experiments of ANDV-, HTNV-, SEOV-, PUUV-, PHV- and TULV-infected HUVEC after exposure to staurosporine (2 μM) for approximately 4 hours. Cells were infected at a low MOI (0.01) to achieve 20 to 30% infection at 4 days post-infection. Following staurosporine-treatment, cells were stained with TUNEL (red) to assess apoptosis, convalescent PUUV patient serum (green) to detect virus infection and DAPI (blue) for nuclear counterstaining. Scale bar, 20 μm. ( b ) Graphs showing TUNEL-positive uninfected and infected cells. Data shown represent the mean ± SD of three independent experiments. ( c ) Western blot analyses of PARP (full-length and cleaved) in total cell lysate from infected and uninfected cells exposed to staurosporine (2 μM) for approximately 4 hours or until observing the effects of staurosporine in treated control cells. MOI 1 of virus was used, resulting in ≥95% of the cells being infected 4 days after infection (Supplementary Fig. 1 ). Specific monoclonal antibodies (mabs 1C12 for ANDV, HTNV, SEOV, PUUV and TULV, and 7B3F7 for PHV) were used for detection of N protein. Calnexin was used as loading control. One representative experiment out of three independent experiments is shown. Band intensity was analysed by densitometry of caspase-3 cleaved PARP and full-length PARP. The ratios in intensity between cleaved and full-length PARP were calculated and compared between infected and uninfected conditions. The ratio from uninfected cells represents maximal caspase-3 cleaved PARP/full-length PARP ratio (100%). The graphs show mean ± SD of the ratio between cleaved and full-length PARP from three experiments. Paired t-test: *p

    Article Snippet: Virus nucleocapsid protein cleavage assay Viruses, heat-inactivated at 96 °C for 10 minutes, were incubated with active human recombinant caspase-3 (with or without caspase-3 inhibitor) or active human recombinant granzyme B, in reaction buffer (BioVision) at 37 °C for 1 hour or as stated in the text.

    Techniques: Immunofluorescence, Infection, Staining, TUNEL Assay, Western Blot

    Orthohantavirus infection hampers the processing of caspase-3 into its active form and inhibits cellular caspase-3 activity. ( a ) Western blot analyses of caspase-3 in total cell lysate from staurosporine-exposed infected and uninfected HUVEC. MOI 1 of virus was used, and cells were treated with 2 μM staurosporine for approximately 4 hours. N protein was detected using the monoclonal antibodies 1C12 for ANDV, HTNV, SEOV, PUUV and TULV, and 7B3F7 for PHV. One representative experiment out of three is shown. Band intensity was analysed by densitometry of cleaved caspase-3 and total caspase-3. A ratio was then calculated and compared between infected and uninfected conditions. Staurosporine-exposed uninfected cells represent maximal cleaved caspase-3/full-length caspase-3 ratio. The graphs show mean ± SD of three independent experiments. ( b ) Caspase-3 activity in infected A549 cells after exposure to 2 μM staurosporine for 4 hours. ≥95% of the A549 cells were infected as assessed by immunofluorescence (Supplementary Fig. 1 ). Cellular caspase-3 activity in infected cells was compared to that in uninfected cells at the same time point. Total protein levels in the samples were measured using a Bradford assay. Staurosporine-treated uninfected cells represent maximal caspase-3 activity/mg of total cellular protein. Results show mean ± SD of three independent experiments. Paired t-test: *p

    Journal: Scientific Reports

    Article Title: Orthohantaviruses belonging to three phylogroups all inhibit apoptosis in infected target cells

    doi: 10.1038/s41598-018-37446-1

    Figure Lengend Snippet: Orthohantavirus infection hampers the processing of caspase-3 into its active form and inhibits cellular caspase-3 activity. ( a ) Western blot analyses of caspase-3 in total cell lysate from staurosporine-exposed infected and uninfected HUVEC. MOI 1 of virus was used, and cells were treated with 2 μM staurosporine for approximately 4 hours. N protein was detected using the monoclonal antibodies 1C12 for ANDV, HTNV, SEOV, PUUV and TULV, and 7B3F7 for PHV. One representative experiment out of three is shown. Band intensity was analysed by densitometry of cleaved caspase-3 and total caspase-3. A ratio was then calculated and compared between infected and uninfected conditions. Staurosporine-exposed uninfected cells represent maximal cleaved caspase-3/full-length caspase-3 ratio. The graphs show mean ± SD of three independent experiments. ( b ) Caspase-3 activity in infected A549 cells after exposure to 2 μM staurosporine for 4 hours. ≥95% of the A549 cells were infected as assessed by immunofluorescence (Supplementary Fig. 1 ). Cellular caspase-3 activity in infected cells was compared to that in uninfected cells at the same time point. Total protein levels in the samples were measured using a Bradford assay. Staurosporine-treated uninfected cells represent maximal caspase-3 activity/mg of total cellular protein. Results show mean ± SD of three independent experiments. Paired t-test: *p

    Article Snippet: Virus nucleocapsid protein cleavage assay Viruses, heat-inactivated at 96 °C for 10 minutes, were incubated with active human recombinant caspase-3 (with or without caspase-3 inhibitor) or active human recombinant granzyme B, in reaction buffer (BioVision) at 37 °C for 1 hour or as stated in the text.

    Techniques: Infection, Activity Assay, Western Blot, Immunofluorescence, Bradford Assay

    ANDV nucleocapsid protein inhibits caspase 3-activity. Analysis of possible interactions between ANDV nucleocapsid protein and caspase 3. ( A ) Western blot analyses showing a cleaved fragment of the ANDV nucleocapsid protein (ANDV-N) after incubation with recombinant caspase 3 in the presence or absence of the caspase 3-inhibitor DEVD-CHO. Full-length and cleaved ANDV-N was visualized with the mAb 7B3/F7. One representative experiment out of three is shown. ( B ) ANDV-infected A549 cells were left untreated or treated with staurosporine, in the presence or absence of the caspase-inhibitor Z-VAD-fmk. Lysed cells were then subjected to Western blot analyses to visualize cleavage of the viral ANDV nucleocapsid protein (ANDV-N) after staurosporine-treatment. Full-length and cleaved ANDV-N was visualized with the mAb 7B3/F7. One representative experiment out of three is shown. ( C ) Caspase 3-activity was measured after pre-incubation of recombinant caspase 3 with recombinant ANDV nucleocapsid protein (rANDV-N) or with control protein (rDHFR) for 30 minutes. Level of caspase 3-activity after co-incubation with rDHFRS represent maximal caspase 3 activity. Level of caspase 3-activity after co-incubation of caspase 3 with rANDV-N was compared with caspase 3-activity after co-incubation with rDHFRS. Data shown are mean ± SEM of three independent experiments carried out in duplicate. Two-tailed Student's t test was used for statistical evaluation; * p

    Journal: PLoS Pathogens

    Article Title: Hantavirus-infection Confers Resistance to Cytotoxic Lymphocyte-Mediated Apoptosis

    doi: 10.1371/journal.ppat.1003272

    Figure Lengend Snippet: ANDV nucleocapsid protein inhibits caspase 3-activity. Analysis of possible interactions between ANDV nucleocapsid protein and caspase 3. ( A ) Western blot analyses showing a cleaved fragment of the ANDV nucleocapsid protein (ANDV-N) after incubation with recombinant caspase 3 in the presence or absence of the caspase 3-inhibitor DEVD-CHO. Full-length and cleaved ANDV-N was visualized with the mAb 7B3/F7. One representative experiment out of three is shown. ( B ) ANDV-infected A549 cells were left untreated or treated with staurosporine, in the presence or absence of the caspase-inhibitor Z-VAD-fmk. Lysed cells were then subjected to Western blot analyses to visualize cleavage of the viral ANDV nucleocapsid protein (ANDV-N) after staurosporine-treatment. Full-length and cleaved ANDV-N was visualized with the mAb 7B3/F7. One representative experiment out of three is shown. ( C ) Caspase 3-activity was measured after pre-incubation of recombinant caspase 3 with recombinant ANDV nucleocapsid protein (rANDV-N) or with control protein (rDHFR) for 30 minutes. Level of caspase 3-activity after co-incubation with rDHFRS represent maximal caspase 3 activity. Level of caspase 3-activity after co-incubation of caspase 3 with rANDV-N was compared with caspase 3-activity after co-incubation with rDHFRS. Data shown are mean ± SEM of three independent experiments carried out in duplicate. Two-tailed Student's t test was used for statistical evaluation; * p

    Article Snippet: Nucleocapsid protein cleavage assays Heat-inactivated ANDV (10 minutes at 96°C) was incubated with either active recombinant human caspase 3 or granzyme B in reaction buffer (BioVision) at 37°C overnight and then subjected to immunoblotting for analysis of cleaved nucleocapsid protein.

    Techniques: Activity Assay, Western Blot, Incubation, Recombinant, Infection, Two Tailed Test

    Hantavirus-infection inhibits NK cell-mediated activation of caspase 3 in endothelial cells. Prevention of caspase 3 activation in infected cells exposed to IL-2-activated NK cells. ( A ) Representative flow cytometry histogram showing cellular caspase 3-activity in uninfected and HTNV-infected HLA class I blocked endothelial cells with and without co-incubation with IL-2-activated NK cells. Data shown is one representative donor out of six. ( B ) Percentage of caspase 3-positive uninfected and HTNV-infected endothelial cells after co-incubation with IL-2-activated NK cells analyzed by flow cytometry. Data shown represent two independent experiments from six donors. Two-tailed Student's t test was used for statistical evaluation; ** p

    Journal: PLoS Pathogens

    Article Title: Hantavirus-infection Confers Resistance to Cytotoxic Lymphocyte-Mediated Apoptosis

    doi: 10.1371/journal.ppat.1003272

    Figure Lengend Snippet: Hantavirus-infection inhibits NK cell-mediated activation of caspase 3 in endothelial cells. Prevention of caspase 3 activation in infected cells exposed to IL-2-activated NK cells. ( A ) Representative flow cytometry histogram showing cellular caspase 3-activity in uninfected and HTNV-infected HLA class I blocked endothelial cells with and without co-incubation with IL-2-activated NK cells. Data shown is one representative donor out of six. ( B ) Percentage of caspase 3-positive uninfected and HTNV-infected endothelial cells after co-incubation with IL-2-activated NK cells analyzed by flow cytometry. Data shown represent two independent experiments from six donors. Two-tailed Student's t test was used for statistical evaluation; ** p

    Article Snippet: Nucleocapsid protein cleavage assays Heat-inactivated ANDV (10 minutes at 96°C) was incubated with either active recombinant human caspase 3 or granzyme B in reaction buffer (BioVision) at 37°C overnight and then subjected to immunoblotting for analysis of cleaved nucleocapsid protein.

    Techniques: Infection, Activation Assay, Flow Cytometry, Cytometry, Activity Assay, Incubation, Two Tailed Test

    Expression of pro-apoptotic active caspase -3 in the chorion

    Journal: International journal of clinical medicine

    Article Title: Increased Apoptosis in Chorionic Trophoblasts of Human Fetal Membranes with Labor at Term

    doi: 10.4236/ijcm.2012.32027

    Figure Lengend Snippet: Expression of pro-apoptotic active caspase -3 in the chorion

    Article Snippet: 30 μg protein from each sample (amnion and choriodecidua)and 2 μL of recombinant human active Caspase-3 (positive control)(Biovision, Mountain View, CA) were loaded onto 12% SDS-PAGE gel.After electrophoretic separation, the protein blots were transferred ontonitrocellulose membranes, and then incubated over-night at 4°C forblocking with 5% nonfat milk in PBS-Tween containing 0.1 %Tween-20.

    Techniques: Expressing

    CDDP-induced rictor downregulation in CDDP-sensitive cells involves caspase-3-mediated cleavage. OV2008 and A2780s were pretreated with Z-VAD FMK (10 µM) and Z-DEVD FMK (50 µM) for 30 minutes before and during CDDP challenge (0-10 µM; 24 h) and rictor content and apoptosis were assessed. OV2008 cells treated with CDDP alone exhibited an intact rictor (200 kDa) and two cleaved products (160 kDa and 130 kDa). CDDP decreased intact rictor content and 160 kDa protein but markedly increased levels of the 130 kDa band. Treatment of A2780s with CDDP resulted in down-regulation of intact rictor but increased the level of the 160 kDa protein and had no effect on the 130 kDa protein. Pre-treatment of the cells with the pan-caspase inhibitor (Z-VAD) or the specific caspase-3 inhibitor (Z-DEVD) significantly attenuated the CDDP-induced changes in intact and cleaved rictor contents in both sensitive cells, and CDDP-induced apoptosis was significantly but not completely attenuated by the presence of the inhibitors in both chemosensitive cell lines. Rictor content was normalized against GAPDH (loading control). Results are presented as mean ± SEM (n=3 and n=5 in OV2008 and in A2780s, respectively). *p

    Journal: PLoS ONE

    Article Title: The mTORC2 Component Rictor Contributes to Cisplatin Resistance in Human Ovarian Cancer Cells

    doi: 10.1371/journal.pone.0075455

    Figure Lengend Snippet: CDDP-induced rictor downregulation in CDDP-sensitive cells involves caspase-3-mediated cleavage. OV2008 and A2780s were pretreated with Z-VAD FMK (10 µM) and Z-DEVD FMK (50 µM) for 30 minutes before and during CDDP challenge (0-10 µM; 24 h) and rictor content and apoptosis were assessed. OV2008 cells treated with CDDP alone exhibited an intact rictor (200 kDa) and two cleaved products (160 kDa and 130 kDa). CDDP decreased intact rictor content and 160 kDa protein but markedly increased levels of the 130 kDa band. Treatment of A2780s with CDDP resulted in down-regulation of intact rictor but increased the level of the 160 kDa protein and had no effect on the 130 kDa protein. Pre-treatment of the cells with the pan-caspase inhibitor (Z-VAD) or the specific caspase-3 inhibitor (Z-DEVD) significantly attenuated the CDDP-induced changes in intact and cleaved rictor contents in both sensitive cells, and CDDP-induced apoptosis was significantly but not completely attenuated by the presence of the inhibitors in both chemosensitive cell lines. Rictor content was normalized against GAPDH (loading control). Results are presented as mean ± SEM (n=3 and n=5 in OV2008 and in A2780s, respectively). *p

    Article Snippet: Rictor and GAPDH RNA primer were from Bioneer (Daejeon, Korea) and human recombinant active caspase 3 was from BioVision (Mountain View, CA, USA).

    Techniques:

    A hypothetical model illustrating the role of rictor in regulation of CDDP sensitivity in OVCA cells. In chemosensitive cells, CDDP activates caspase-3 and induces proteasomal degradation for rictor processing, and consequently destabilizes mTORC2, The unstable mTORC2 complex then facilitates Akt phosphorylation at Ser473, an event known to promote Akt proteasomal degradation and the induction of apoptosis. However, in chemoresistant cells, high level of stabilized rictor promotes Akt activation and stabilization, thereby contributing to CDDP resistance.

    Journal: PLoS ONE

    Article Title: The mTORC2 Component Rictor Contributes to Cisplatin Resistance in Human Ovarian Cancer Cells

    doi: 10.1371/journal.pone.0075455

    Figure Lengend Snippet: A hypothetical model illustrating the role of rictor in regulation of CDDP sensitivity in OVCA cells. In chemosensitive cells, CDDP activates caspase-3 and induces proteasomal degradation for rictor processing, and consequently destabilizes mTORC2, The unstable mTORC2 complex then facilitates Akt phosphorylation at Ser473, an event known to promote Akt proteasomal degradation and the induction of apoptosis. However, in chemoresistant cells, high level of stabilized rictor promotes Akt activation and stabilization, thereby contributing to CDDP resistance.

    Article Snippet: Rictor and GAPDH RNA primer were from Bioneer (Daejeon, Korea) and human recombinant active caspase 3 was from BioVision (Mountain View, CA, USA).

    Techniques: Activation Assay

    Proteasome-mediated degradation and caspase-3 activity are responsible for CDDP-induced rictor down-regulation in chemosensitive OVCA. A . OV2008 cells were pretreated (30 min) with the proteasomal inhibitors [epoxomycin (10 nM) and lacytasystin (4 µM)] and subjected to CDDP challenge (0-10 µM; 24 h). Rictor content and apoptosis were analyzed by Western blotting and nuclear morphology assessment. Both epoxomicin and lactacystin effectively blocked CDDP-induced rictor degradation (P

    Journal: PLoS ONE

    Article Title: The mTORC2 Component Rictor Contributes to Cisplatin Resistance in Human Ovarian Cancer Cells

    doi: 10.1371/journal.pone.0075455

    Figure Lengend Snippet: Proteasome-mediated degradation and caspase-3 activity are responsible for CDDP-induced rictor down-regulation in chemosensitive OVCA. A . OV2008 cells were pretreated (30 min) with the proteasomal inhibitors [epoxomycin (10 nM) and lacytasystin (4 µM)] and subjected to CDDP challenge (0-10 µM; 24 h). Rictor content and apoptosis were analyzed by Western blotting and nuclear morphology assessment. Both epoxomicin and lactacystin effectively blocked CDDP-induced rictor degradation (P

    Article Snippet: Rictor and GAPDH RNA primer were from Bioneer (Daejeon, Korea) and human recombinant active caspase 3 was from BioVision (Mountain View, CA, USA).

    Techniques: Activity Assay, Western Blot

    FKBP38 interacts with the flexible loop of Bcl-2-contained caspase cleavage site. A , 2 μg of free Bcl-2ΔTM- or His-FKBP38-bound Bcl-2ΔTM were incubated with 50 ng of recombinant human active caspase-3/CPP32. The proteins were resolved by 12% SDS-PAGE gel and Bcl-2ΔTM, and proteolyzed fragments were detected with anti-Bcl-2 antibody. B , cell lysates from Huh7 cells transiently overexpressing FLAG-FKBP38 with YFP-Bcl-2 full-length, YFP-Bcl-2ΔTM, or YFP-Bcl-2ΔLΔTM were immunoprecipitated with anti-FLAG antibody. The cell lysates (1/20 of those used in immunoprecipitation ( IP )) and the immune complexes were then analyzed by immunoblotting with anti-GFP or anti-FLAG antibodies. C , shown is FRET between Bcl-2 with FKBP38. Cells were seeded on a coverslip inside a six-well plate and co-transfected with plasmid encoding FKBP38 (pECFP-FKBP38) together with constructs (pEYFP-Bcl-2, pEYFP-Bcl-2ΔTM, or pEYFP-Bcl-2ΔLΔTM). 24 h later a FRET assay were performed as described under “Experimental Procedures.” Data represent three independent experiments. D , cell lysates from Huh7 cells transiently overexpressing FLAG-FKBP38 with YFP-Bcl-2 full-length or YFP-Bcl-2 D34A were immunoblotted with anti-FLAG, anti-GFP, and anti-actin antibodies. WT , wild type.

    Journal: The Journal of Biological Chemistry

    Article Title: FKBP38 Protects Bcl-2 from Caspase-dependent Degradation *

    doi: 10.1074/jbc.M109.032466

    Figure Lengend Snippet: FKBP38 interacts with the flexible loop of Bcl-2-contained caspase cleavage site. A , 2 μg of free Bcl-2ΔTM- or His-FKBP38-bound Bcl-2ΔTM were incubated with 50 ng of recombinant human active caspase-3/CPP32. The proteins were resolved by 12% SDS-PAGE gel and Bcl-2ΔTM, and proteolyzed fragments were detected with anti-Bcl-2 antibody. B , cell lysates from Huh7 cells transiently overexpressing FLAG-FKBP38 with YFP-Bcl-2 full-length, YFP-Bcl-2ΔTM, or YFP-Bcl-2ΔLΔTM were immunoprecipitated with anti-FLAG antibody. The cell lysates (1/20 of those used in immunoprecipitation ( IP )) and the immune complexes were then analyzed by immunoblotting with anti-GFP or anti-FLAG antibodies. C , shown is FRET between Bcl-2 with FKBP38. Cells were seeded on a coverslip inside a six-well plate and co-transfected with plasmid encoding FKBP38 (pECFP-FKBP38) together with constructs (pEYFP-Bcl-2, pEYFP-Bcl-2ΔTM, or pEYFP-Bcl-2ΔLΔTM). 24 h later a FRET assay were performed as described under “Experimental Procedures.” Data represent three independent experiments. D , cell lysates from Huh7 cells transiently overexpressing FLAG-FKBP38 with YFP-Bcl-2 full-length or YFP-Bcl-2 D34A were immunoblotted with anti-FLAG, anti-GFP, and anti-actin antibodies. WT , wild type.

    Article Snippet: 2 μg of Bcl-2ΔTM or His-FKBP38-bound Bcl-2ΔTM were incubated with 50 ng of recombinant human active caspase-3/CPP32 (BioVision, Mountain View, CA) in a 30-μl reaction buffer containing 50 m m HEPES, pH 7.4, 100 m m NaCl, 10 m m dithiothreitol, 10% sucrose, and 0.1% CHAPS for 16 h at 37 °C.

    Techniques: Incubation, Recombinant, SDS Page, Immunoprecipitation, Transfection, Plasmid Preparation, Construct

    Fig. 3. GrB induces DNA fragmentation in the absence of caspase-3 activation. ( A ) Structures of the caspase-3/-7-specific inhibitor (compound 1) and a 600-fold less active analog (compound 2). ( B ) Compound 1 blocks Fas-mediated apoptosis in a dose-dependent fashion. Jurkat cells were pre-treated with different concentrations of compound 1 (lanes 7–12) or compound 2 (lanes 1–6). Apoptosis was induced by cross-linking cell surface Fas receptors using mAb to Fas (M3, 20 µg/ml). Abrogation of Fas-induced apoptosis by compound 1 was monitored by DNA fragmentation, annexin V externalization, caspase-3 cleavage and DFF processing. No effect was noticed for compound 2 even at the highest concentration used (lanes 1–6). ( C ) DFF cleavage and DNA fragmentation occur in the absence of caspase-3 activation. Jurkat cells (1 × 10 6 cells per well) were pre-treated with 75 µM compound 1 or compound 2. Apoptosis by GrB/Ad 2 was induced as described in Materials and methods. Cell death was assayed as in (B). Pre-treatment of the cells with the caspase-3-specific inhibitor compound 1 did not prevent DFF processing and DNA fragmentation induced by GrB (lane 6). Pre-treatment of cells with the caspase-3-specific inhibitor compound 1 (lane 6), but not its analog compound 2 (lane 5), or DMSO alone (lane 4) resulted in a significant decrease in annexin V externalization. Untreated Jurkat cells (lane 1) and Jurkat cells treated with GrB or Ad 2 alone (lanes 2 and 3, respectively) showed no significant sign of apoptosis. These results are representative of two separate experiments. ( D ) Jurkat cells (1 × 10 5 cells per well) were pre-treated with 75 µM compound 1 or compound 2, and apoptosis was induced by recombinant GrB/perforin as described in Materials and methods. Cell death was assayed by 51 Cr release. Pre-treatment with compound 1, but not compound 2, or DMSO blocked further processing of caspase-3 to the p17 active form (lanes 6, 4 and 5, respectively, upper panel) and processing of caspase-mediated cleavage of Rho-GDI substrate (lanes 6, 4 and 5, respectively, lower panel). Blockage of caspase-3 activity had no effect on DFF processing (lane 6, middle panel). Untreated Jurkat cells (lane 1) and those treated with perforin or GrB alone (lanes 2 and 3, respectively) showed no significant sign of apoptosis. These results are representative of three separate experiments.

    Journal: The EMBO Journal

    Article Title: Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation

    doi: 10.1093/emboj/20.12.3101

    Figure Lengend Snippet: Fig. 3. GrB induces DNA fragmentation in the absence of caspase-3 activation. ( A ) Structures of the caspase-3/-7-specific inhibitor (compound 1) and a 600-fold less active analog (compound 2). ( B ) Compound 1 blocks Fas-mediated apoptosis in a dose-dependent fashion. Jurkat cells were pre-treated with different concentrations of compound 1 (lanes 7–12) or compound 2 (lanes 1–6). Apoptosis was induced by cross-linking cell surface Fas receptors using mAb to Fas (M3, 20 µg/ml). Abrogation of Fas-induced apoptosis by compound 1 was monitored by DNA fragmentation, annexin V externalization, caspase-3 cleavage and DFF processing. No effect was noticed for compound 2 even at the highest concentration used (lanes 1–6). ( C ) DFF cleavage and DNA fragmentation occur in the absence of caspase-3 activation. Jurkat cells (1 × 10 6 cells per well) were pre-treated with 75 µM compound 1 or compound 2. Apoptosis by GrB/Ad 2 was induced as described in Materials and methods. Cell death was assayed as in (B). Pre-treatment of the cells with the caspase-3-specific inhibitor compound 1 did not prevent DFF processing and DNA fragmentation induced by GrB (lane 6). Pre-treatment of cells with the caspase-3-specific inhibitor compound 1 (lane 6), but not its analog compound 2 (lane 5), or DMSO alone (lane 4) resulted in a significant decrease in annexin V externalization. Untreated Jurkat cells (lane 1) and Jurkat cells treated with GrB or Ad 2 alone (lanes 2 and 3, respectively) showed no significant sign of apoptosis. These results are representative of two separate experiments. ( D ) Jurkat cells (1 × 10 5 cells per well) were pre-treated with 75 µM compound 1 or compound 2, and apoptosis was induced by recombinant GrB/perforin as described in Materials and methods. Cell death was assayed by 51 Cr release. Pre-treatment with compound 1, but not compound 2, or DMSO blocked further processing of caspase-3 to the p17 active form (lanes 6, 4 and 5, respectively, upper panel) and processing of caspase-mediated cleavage of Rho-GDI substrate (lanes 6, 4 and 5, respectively, lower panel). Blockage of caspase-3 activity had no effect on DFF processing (lane 6, middle panel). Untreated Jurkat cells (lane 1) and those treated with perforin or GrB alone (lanes 2 and 3, respectively) showed no significant sign of apoptosis. These results are representative of three separate experiments.

    Article Snippet: [ ] Mitamura S., Ikawa,H., Mizuno,N., Kaziro,Y. and Itoh,H. (1998) Cytosolic nuclease activated by caspase-3 and inhibited by DFF-45.

    Techniques: Activation Assay, Concentration Assay, Recombinant, Activity Assay

    Fig. 2. GrB shares the DETD 117 ↓ cleavage site with caspase-3 and exhibits an additional specificity at VTGD 6 ↓ on DFF45. ( A ) Schematic diagram of wild-type DFF45 (DFF45-WT) and caspase-3 cleavage site mutants (DFF45-M1, -M2, -M12). Overlap PCR was used to mutate aspartic acid (D) residues of the first (D 117 ), second (D 224 ) or both caspase-3 cleavage sites to glutamic acid (E). Caspase-3 cleavage sites are indicated. ( B ) Radiolabeled His-tagged DFF45 proteins were incubated in the presence of 1 U of GrB (lanes 1–4) or 10 U of caspase-3 (lanes 5–8) for the indicated time intervals, and the reactions were analyzed by autoradiography. Incubation of DFF45-WT or its mutants with GrB or caspase-3 showed that GrB shares the D 117 cleavage site with caspase-3 and exhibits additional proteolytic activity. These results are representative of three separate experiments. ( C ) Identification of the additional GrB cleavage site on DFF45. cDNAs representing DFF45 fragments (DFF45-F1, -F2 and -F3 corresponding to amino acids 1–117, 118–224 and 225–334, respectively) were cloned, and radiolabeled proteins were generated as described in Materials and methods. ( D ) Radiolabeled proteins were treated as in (B). Only DFF45-F1, corresponding to the N-terminus (1–117), was cleaved by GrB. These results are representative of two separate experiments.

    Journal: The EMBO Journal

    Article Title: Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation

    doi: 10.1093/emboj/20.12.3101

    Figure Lengend Snippet: Fig. 2. GrB shares the DETD 117 ↓ cleavage site with caspase-3 and exhibits an additional specificity at VTGD 6 ↓ on DFF45. ( A ) Schematic diagram of wild-type DFF45 (DFF45-WT) and caspase-3 cleavage site mutants (DFF45-M1, -M2, -M12). Overlap PCR was used to mutate aspartic acid (D) residues of the first (D 117 ), second (D 224 ) or both caspase-3 cleavage sites to glutamic acid (E). Caspase-3 cleavage sites are indicated. ( B ) Radiolabeled His-tagged DFF45 proteins were incubated in the presence of 1 U of GrB (lanes 1–4) or 10 U of caspase-3 (lanes 5–8) for the indicated time intervals, and the reactions were analyzed by autoradiography. Incubation of DFF45-WT or its mutants with GrB or caspase-3 showed that GrB shares the D 117 cleavage site with caspase-3 and exhibits additional proteolytic activity. These results are representative of three separate experiments. ( C ) Identification of the additional GrB cleavage site on DFF45. cDNAs representing DFF45 fragments (DFF45-F1, -F2 and -F3 corresponding to amino acids 1–117, 118–224 and 225–334, respectively) were cloned, and radiolabeled proteins were generated as described in Materials and methods. ( D ) Radiolabeled proteins were treated as in (B). Only DFF45-F1, corresponding to the N-terminus (1–117), was cleaved by GrB. These results are representative of two separate experiments.

    Article Snippet: [ ] Mitamura S., Ikawa,H., Mizuno,N., Kaziro,Y. and Itoh,H. (1998) Cytosolic nuclease activated by caspase-3 and inhibited by DFF-45.

    Techniques: Polymerase Chain Reaction, Incubation, Autoradiography, Activity Assay, Clone Assay, Generated

    Fig. 1. Nuclear compartmentalization and efficient in vitro processing of DFF45 protein by GrB. ( A ) Double labeling confocal immunofluorescence microscopy of Jurkat cells. Jurkat cells were stained by anti-β-actin monoclonal antibodies and FITC-coupled anti-mouse IgG. DFF45 and DFF40 were detected by using either anti-DFF45 or anti-DFF40 rabbit polyclonal antibodies, respectively, and TRITC-coupled anti-rabbit IgG as described in Materials and methods. Controls were performed by labeling cells with either the secondary TRITC-coupled anti-rabbit IgG alone, or with pre-immune rabbit serum. Each picture is representative of at least 10 fields of two independent experiments at a magnification of 1000×. ( B ) Time-dependent cleavage of DFF45 by GrB. rDFF45 (12 ng) was incubated for the times indicated with 1 U of GrB (lanes 1–9) or with 10 U of caspase-3 (lanes 10–18) at 37°C. Reactions were stopped by adding Laemmli sample buffer and analyzed by western blotting with anti-DFF45 Ab. Results are representative of three separate experiments. ( C ) Dose-dependent cleavage of DFF45 by GrB. Purified rDFF45 (12 ng) was incubated with different concentrations of GrB at 37°C for 20 min and subjected to western blot analysis using anti-DFF45 antiserum.

    Journal: The EMBO Journal

    Article Title: Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation

    doi: 10.1093/emboj/20.12.3101

    Figure Lengend Snippet: Fig. 1. Nuclear compartmentalization and efficient in vitro processing of DFF45 protein by GrB. ( A ) Double labeling confocal immunofluorescence microscopy of Jurkat cells. Jurkat cells were stained by anti-β-actin monoclonal antibodies and FITC-coupled anti-mouse IgG. DFF45 and DFF40 were detected by using either anti-DFF45 or anti-DFF40 rabbit polyclonal antibodies, respectively, and TRITC-coupled anti-rabbit IgG as described in Materials and methods. Controls were performed by labeling cells with either the secondary TRITC-coupled anti-rabbit IgG alone, or with pre-immune rabbit serum. Each picture is representative of at least 10 fields of two independent experiments at a magnification of 1000×. ( B ) Time-dependent cleavage of DFF45 by GrB. rDFF45 (12 ng) was incubated for the times indicated with 1 U of GrB (lanes 1–9) or with 10 U of caspase-3 (lanes 10–18) at 37°C. Reactions were stopped by adding Laemmli sample buffer and analyzed by western blotting with anti-DFF45 Ab. Results are representative of three separate experiments. ( C ) Dose-dependent cleavage of DFF45 by GrB. Purified rDFF45 (12 ng) was incubated with different concentrations of GrB at 37°C for 20 min and subjected to western blot analysis using anti-DFF45 antiserum.

    Article Snippet: [ ] Mitamura S., Ikawa,H., Mizuno,N., Kaziro,Y. and Itoh,H. (1998) Cytosolic nuclease activated by caspase-3 and inhibited by DFF-45.

    Techniques: In Vitro, Labeling, Immunofluorescence, Microscopy, Staining, Incubation, Western Blot, Purification

    Fig. 4. Cleavage of DFF45 at residue D 117 is necessary and sufficient to induce DNA fragmentation. ( A ) Jurkat cells were stably transfected with SRα Neo/DFF45-WT, -M1, -M2 or -M12 (lanes 2–5, respectively). The empty SRα Neo vector was transfected in Jurkat cells and used as a control (lane 1). Expression of the transfected (exogenous) and native (endogenous) DFF45 is indicated. ( B ) Stably transfected Jurkat cells were triggered to undergo apoptosis by either Fas (lanes 2–6) or GrB/Ad 2 (lanes 8–12) for 5 h as described in Materials and methods. Following the reaction, fragmented DNA was extracted and analyzed on a 2% agarose gel. Mutation of residue D 117 completely abolished DNA fragmentation following GrB- (lane 10) or Fas (lane 4)-induced apoptosis. The results are representative of three separate experiments. ( C ) Direct cleavage of DFF45 by GrB triggers CAD nuclease activity in a cell-free system. The human CAD was expressed in the in vitro transcription and translation system in the absence (lanes 3 and 7) or presence (lanes 4 and 8) of 160 ng of rDFF45 as described in Materials and methods. CAD nuclease activity using plasmid DNA as a substrate was determined in the presence of 0.12 µM GrB (lanes 5–8) or 0.2 µM caspase-3 (lanes 1–4). Samples containing plasmid alone (lanes 1 and 5) or rDFF45 alone (lanes 2 and 6) were subjected to a similar procedure in the presence of GrB (lanes 5 and 6) or caspase-3 (lanes 1 and 2). The results are representative of two separate experiments.

    Journal: The EMBO Journal

    Article Title: Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation

    doi: 10.1093/emboj/20.12.3101

    Figure Lengend Snippet: Fig. 4. Cleavage of DFF45 at residue D 117 is necessary and sufficient to induce DNA fragmentation. ( A ) Jurkat cells were stably transfected with SRα Neo/DFF45-WT, -M1, -M2 or -M12 (lanes 2–5, respectively). The empty SRα Neo vector was transfected in Jurkat cells and used as a control (lane 1). Expression of the transfected (exogenous) and native (endogenous) DFF45 is indicated. ( B ) Stably transfected Jurkat cells were triggered to undergo apoptosis by either Fas (lanes 2–6) or GrB/Ad 2 (lanes 8–12) for 5 h as described in Materials and methods. Following the reaction, fragmented DNA was extracted and analyzed on a 2% agarose gel. Mutation of residue D 117 completely abolished DNA fragmentation following GrB- (lane 10) or Fas (lane 4)-induced apoptosis. The results are representative of three separate experiments. ( C ) Direct cleavage of DFF45 by GrB triggers CAD nuclease activity in a cell-free system. The human CAD was expressed in the in vitro transcription and translation system in the absence (lanes 3 and 7) or presence (lanes 4 and 8) of 160 ng of rDFF45 as described in Materials and methods. CAD nuclease activity using plasmid DNA as a substrate was determined in the presence of 0.12 µM GrB (lanes 5–8) or 0.2 µM caspase-3 (lanes 1–4). Samples containing plasmid alone (lanes 1 and 5) or rDFF45 alone (lanes 2 and 6) were subjected to a similar procedure in the presence of GrB (lanes 5 and 6) or caspase-3 (lanes 1 and 2). The results are representative of two separate experiments.

    Article Snippet: [ ] Mitamura S., Ikawa,H., Mizuno,N., Kaziro,Y. and Itoh,H. (1998) Cytosolic nuclease activated by caspase-3 and inhibited by DFF-45.

    Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Agarose Gel Electrophoresis, Mutagenesis, Activity Assay, In Vitro

    Fig. 5. Mechanism of GrB-induced DNA fragmentation in the absence of caspase-3 activity. Following the induction of cellular cytotoxicity, GrB enters target cells and becomes activated in the presence of perforin. Rapid translocation of GrB from the cytoplasm to the nucleus leads to nuclear substrate cleavage. Direct cleavage of DFF45 by GrB induces DNA fragmentation via bypassing of caspase activation. GrB-induced caspase activity will serve to augment the apoptotic process.

    Journal: The EMBO Journal

    Article Title: Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation

    doi: 10.1093/emboj/20.12.3101

    Figure Lengend Snippet: Fig. 5. Mechanism of GrB-induced DNA fragmentation in the absence of caspase-3 activity. Following the induction of cellular cytotoxicity, GrB enters target cells and becomes activated in the presence of perforin. Rapid translocation of GrB from the cytoplasm to the nucleus leads to nuclear substrate cleavage. Direct cleavage of DFF45 by GrB induces DNA fragmentation via bypassing of caspase activation. GrB-induced caspase activity will serve to augment the apoptotic process.

    Article Snippet: [ ] Mitamura S., Ikawa,H., Mizuno,N., Kaziro,Y. and Itoh,H. (1998) Cytosolic nuclease activated by caspase-3 and inhibited by DFF-45.

    Techniques: Activity Assay, Translocation Assay, Activation Assay

    Purified and lysate-associated c-gelsolin is cleaved by rec.gzmB independent of caspase 3. A  and  B , rec.c-gelsolin was treated with rec.gzmB in a dose ( A )- and time-dependent manner ( B ).  C , specific proteolysis of c-gelsolin by gzmB was verified using

    Journal: The Journal of Biological Chemistry

    Article Title: Granzyme B-induced and Caspase 3-dependent Cleavage of Gelsolin by Mouse Cytotoxic T Cells Modifies Cytoskeleton Dynamics *

    doi: 10.1074/jbc.M109.056028

    Figure Lengend Snippet: Purified and lysate-associated c-gelsolin is cleaved by rec.gzmB independent of caspase 3. A and B , rec.c-gelsolin was treated with rec.gzmB in a dose ( A )- and time-dependent manner ( B ). C , specific proteolysis of c-gelsolin by gzmB was verified using

    Article Snippet: Rec.caspase 3 was purchased from Axxora GmbH.

    Techniques: Purification

    The cleavage of c-gelsolin in intact cells by purified or Tc cell-associated gzmB is dependent on caspase 3. A , MEF wt cells preincubated with the caspase 3/7-specific inhibitor zDEVD-cmk or the pan-caspase inhibitor zVAD-fmk were treated with rec.gzmB

    Journal: The Journal of Biological Chemistry

    Article Title: Granzyme B-induced and Caspase 3-dependent Cleavage of Gelsolin by Mouse Cytotoxic T Cells Modifies Cytoskeleton Dynamics *

    doi: 10.1074/jbc.M109.056028

    Figure Lengend Snippet: The cleavage of c-gelsolin in intact cells by purified or Tc cell-associated gzmB is dependent on caspase 3. A , MEF wt cells preincubated with the caspase 3/7-specific inhibitor zDEVD-cmk or the pan-caspase inhibitor zVAD-fmk were treated with rec.gzmB

    Article Snippet: Rec.caspase 3 was purchased from Axxora GmbH.

    Techniques: Purification

    Immunofluorescent dopaminergic neurons in primary cultures of rat mesencephalic neurons fixed after 12 hr of 1-μM MPP + treatment stained with a TH monoclonal mouse antibody revealed by FITC ( A ) and a rabbit polyclonal antibody recognizing activated caspase-3 (CM1) revealed by tetramethylrhodamine B isothiocyanate ( B ). Hoechst 33258 staining reveals an intact nucleus in this neuron ( C , arrow). (Bar = 20 μm.) In contrast, in a TH-positive neuron ( D ) with CM1 staining ( E ), DNA condensation assessed by Hoechst 33258 can be detected after 72 hr of MPP + treatment ( F , arrow). Note that a higher magnification was used to show DNA condensation. (Bar = 10 μm.)

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

    Article Title: Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson's disease

    doi:

    Figure Lengend Snippet: Immunofluorescent dopaminergic neurons in primary cultures of rat mesencephalic neurons fixed after 12 hr of 1-μM MPP + treatment stained with a TH monoclonal mouse antibody revealed by FITC ( A ) and a rabbit polyclonal antibody recognizing activated caspase-3 (CM1) revealed by tetramethylrhodamine B isothiocyanate ( B ). Hoechst 33258 staining reveals an intact nucleus in this neuron ( C , arrow). (Bar = 20 μm.) In contrast, in a TH-positive neuron ( D ) with CM1 staining ( E ), DNA condensation assessed by Hoechst 33258 can be detected after 72 hr of MPP + treatment ( F , arrow). Note that a higher magnification was used to show DNA condensation. (Bar = 10 μm.)

    Article Snippet: To positively detect the cleaved p17 fragment of caspase-3 using the CM1 antibody, 15 μg of Jurkat cell lysates was preincubated with 500 ng of human recombinant caspase-3 (PharMingen) for 60 min at 37°C.

    Techniques: Staining

    High-power photomicrograph showing Lewy bodies stained for ubiquitin (single arrow) and caspase-3 (triple arrows) in a SNpc neuromelanin-containing (thick arrow) neuron in a transverse section of PD SNpc. Note that ubiquitin-containing fibers and extraneuronal melanin can be seen. (Bar = 30 μm.)

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

    Article Title: Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson's disease

    doi:

    Figure Lengend Snippet: High-power photomicrograph showing Lewy bodies stained for ubiquitin (single arrow) and caspase-3 (triple arrows) in a SNpc neuromelanin-containing (thick arrow) neuron in a transverse section of PD SNpc. Note that ubiquitin-containing fibers and extraneuronal melanin can be seen. (Bar = 30 μm.)

    Article Snippet: To positively detect the cleaved p17 fragment of caspase-3 using the CM1 antibody, 15 μg of Jurkat cell lysates was preincubated with 500 ng of human recombinant caspase-3 (PharMingen) for 60 min at 37°C.

    Techniques: Staining

    Characterization of caspase-3 staining. ( A ) Specificity of the anti-caspase-3 p20 polyclonal rabbit antibody. Western immunoblot of caspase-3 from 50 μg of SNpc protein extracted from three control and three parkinsonian mesencephalons after SDS/PAGE. Molecular mass markers in the first lane are given in kDa and allow identification of caspase-3 protein at molecular masses of 32 kDa (including prodomain) and 30 kDa (without prodomain). ( B ) High-power photomicrograph showing cytosolic caspase-3 immunostaining of SNpc neuromelanin-containing neurons in transverse sections of control SNpc. ( C and D ) Immunofluorescent dopaminergic neurons in transverse SNpc sections of a control subject stained with a caspase-3 polyclonal rabbit antibody revealed by fluorescein ( C ) and a TH monoclonal mouse antibody revealed by rhodamine 600 ( D ). Whereas caspase-3 immunoreactivity is confined to the cytosol, TH staining can be observed in the perikarya and dendrites. The middle portion of the neuron is occupied by neuromelanin. (Bars = 30 μm.)

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

    Article Title: Caspase-3: A vulnerability factor and final effector in apoptotic death of dopaminergic neurons in Parkinson's disease

    doi:

    Figure Lengend Snippet: Characterization of caspase-3 staining. ( A ) Specificity of the anti-caspase-3 p20 polyclonal rabbit antibody. Western immunoblot of caspase-3 from 50 μg of SNpc protein extracted from three control and three parkinsonian mesencephalons after SDS/PAGE. Molecular mass markers in the first lane are given in kDa and allow identification of caspase-3 protein at molecular masses of 32 kDa (including prodomain) and 30 kDa (without prodomain). ( B ) High-power photomicrograph showing cytosolic caspase-3 immunostaining of SNpc neuromelanin-containing neurons in transverse sections of control SNpc. ( C and D ) Immunofluorescent dopaminergic neurons in transverse SNpc sections of a control subject stained with a caspase-3 polyclonal rabbit antibody revealed by fluorescein ( C ) and a TH monoclonal mouse antibody revealed by rhodamine 600 ( D ). Whereas caspase-3 immunoreactivity is confined to the cytosol, TH staining can be observed in the perikarya and dendrites. The middle portion of the neuron is occupied by neuromelanin. (Bars = 30 μm.)

    Article Snippet: To positively detect the cleaved p17 fragment of caspase-3 using the CM1 antibody, 15 μg of Jurkat cell lysates was preincubated with 500 ng of human recombinant caspase-3 (PharMingen) for 60 min at 37°C.

    Techniques: Staining, Western Blot, SDS Page, Immunostaining

    Inhibition of caspase-3 by zinc. Caspase-3 (1.7 nM) was incubated with various amounts of zinc. Activity was assayed after 5 min.

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

    Article Title: Inhibitory sites in enzymes: Zinc removal and reactivation by thionein

    doi:

    Figure Lengend Snippet: Inhibition of caspase-3 by zinc. Caspase-3 (1.7 nM) was incubated with various amounts of zinc. Activity was assayed after 5 min.

    Article Snippet: Recombinant human caspase-3 from PharMingen was diluted to a final concentration of 1.7 nM in buffer.

    Techniques: Inhibition, Incubation, Activity Assay

    Abolition of caspase-3 cleavage of PARP by mutation at the cleavage site (DEVD). (A) The full-length human PARP (hPARP) cDNA was under control of the T7 and SV40 promoters (PARP/WT). An Asp (D)→Asn (N) transition (codon 214, asterisk) was introduced into the DEVD site, resulting in a mutant construct designated D214N. The polyadenylation signal [(poly (A)] was from SV40. MCS, multiple cloning sites. The arrow indicates the caspase cleavage site. (B) [ 35 S]methionine-labeled PARP was generated by an in vitro coupled transcription-translation system. Translated products from PARP/WT and D214N vectors were incubated either with (+) or without (−) purified human recombinant caspase-3. (C) Western blot analysis of PARP expression and cleavage in transfected fibroblasts. Stably transfected clones expressing wild-type PARP (WT/2 and WT/3) and those expressing mutant PARP (DN/8 and DN/10) were either treated or not treated with TNF-α (20 ng/ml); this was followed by Western blot analysis with polyclonal antiserum Vic-5. Full-length PARP (113 kDa) and two cleaved fragments (89 and 24 kDa) are indicated by arrowheads.

    Journal: Molecular and Cellular Biology

    Article Title: Failure of Poly(ADP-Ribose) Polymerase Cleavage by Caspases Leads to Induction of Necrosis and Enhanced Apoptosis

    doi:

    Figure Lengend Snippet: Abolition of caspase-3 cleavage of PARP by mutation at the cleavage site (DEVD). (A) The full-length human PARP (hPARP) cDNA was under control of the T7 and SV40 promoters (PARP/WT). An Asp (D)→Asn (N) transition (codon 214, asterisk) was introduced into the DEVD site, resulting in a mutant construct designated D214N. The polyadenylation signal [(poly (A)] was from SV40. MCS, multiple cloning sites. The arrow indicates the caspase cleavage site. (B) [ 35 S]methionine-labeled PARP was generated by an in vitro coupled transcription-translation system. Translated products from PARP/WT and D214N vectors were incubated either with (+) or without (−) purified human recombinant caspase-3. (C) Western blot analysis of PARP expression and cleavage in transfected fibroblasts. Stably transfected clones expressing wild-type PARP (WT/2 and WT/3) and those expressing mutant PARP (DN/8 and DN/10) were either treated or not treated with TNF-α (20 ng/ml); this was followed by Western blot analysis with polyclonal antiserum Vic-5. Full-length PARP (113 kDa) and two cleaved fragments (89 and 24 kDa) are indicated by arrowheads.

    Article Snippet: A 1.5-μl portion of the translated product was incubated with 4 U of purified human recombinant caspase-3 (a kind gift of D. Nicholson, Merck Frosst, Pointe Claire-Dorval, Canada) for 1 h at 30°C in a buffer containing 50 mM HEPES-KOH (pH 7), 10% (wt/vol) sucrose, 2 mM EDTA, 0.1% (wt/vol) 3-[(3-cholamidoproplyl)-dimethylammonio]-1-propanesulfonate (CHAPS), and 5 mM dithiothreitol.

    Techniques: Mutagenesis, Construct, Clone Assay, Labeling, Generated, In Vitro, Incubation, Purification, Recombinant, Western Blot, Expressing, Transfection, Stable Transfection

    Characterization of wild-type and caspase-resistant PARP in transfected mouse fibroblast cells. (A) Upper panels, immunofluorescence analysis detects PARP expression in nuclei of WT/2 cells and DN/8 cells with a polyclonal anti-PARP antiserum, Vic-5. Lower panels, following treatment with MNNG, poly(ADP-ribose) polymer formation in WT/2 and DN/8 cells was detected by immunofluorescence after staining with a mouse monoclonal antibody, 10H. A11 cells ( PARP −/− ) were used as a control. (B) Analysis of caspase-3 activity in WT/2, DN/8, and A11 cells which were either treated or not treated with 20 ng of TNF-α per ml for 12 h. Cell lysates were preincubated with or without the caspase-3 inhibitor DEVD-CHO, and caspase-3 activity was measured by using a fluorescent substrate, DEVD-AFC, in a fluorometer. Data are from one of three independent experiments and are expressed as means ± standard deviations for duplicate samples. (C) Expression of caspase-resistant PARP sensitizes fibroblasts to TNF-α-induced cell death. WT/2 and DN/8 cells were either treated or not treated with TNF-α for 12 h, and cell viability was analyzed by the MTT assay. The results are shown as means ± standard deviations for quadruplicate samples. Data are from one of three independent experiments.

    Journal: Molecular and Cellular Biology

    Article Title: Failure of Poly(ADP-Ribose) Polymerase Cleavage by Caspases Leads to Induction of Necrosis and Enhanced Apoptosis

    doi:

    Figure Lengend Snippet: Characterization of wild-type and caspase-resistant PARP in transfected mouse fibroblast cells. (A) Upper panels, immunofluorescence analysis detects PARP expression in nuclei of WT/2 cells and DN/8 cells with a polyclonal anti-PARP antiserum, Vic-5. Lower panels, following treatment with MNNG, poly(ADP-ribose) polymer formation in WT/2 and DN/8 cells was detected by immunofluorescence after staining with a mouse monoclonal antibody, 10H. A11 cells ( PARP −/− ) were used as a control. (B) Analysis of caspase-3 activity in WT/2, DN/8, and A11 cells which were either treated or not treated with 20 ng of TNF-α per ml for 12 h. Cell lysates were preincubated with or without the caspase-3 inhibitor DEVD-CHO, and caspase-3 activity was measured by using a fluorescent substrate, DEVD-AFC, in a fluorometer. Data are from one of three independent experiments and are expressed as means ± standard deviations for duplicate samples. (C) Expression of caspase-resistant PARP sensitizes fibroblasts to TNF-α-induced cell death. WT/2 and DN/8 cells were either treated or not treated with TNF-α for 12 h, and cell viability was analyzed by the MTT assay. The results are shown as means ± standard deviations for quadruplicate samples. Data are from one of three independent experiments.

    Article Snippet: A 1.5-μl portion of the translated product was incubated with 4 U of purified human recombinant caspase-3 (a kind gift of D. Nicholson, Merck Frosst, Pointe Claire-Dorval, Canada) for 1 h at 30°C in a buffer containing 50 mM HEPES-KOH (pH 7), 10% (wt/vol) sucrose, 2 mM EDTA, 0.1% (wt/vol) 3-[(3-cholamidoproplyl)-dimethylammonio]-1-propanesulfonate (CHAPS), and 5 mM dithiothreitol.

    Techniques: Transfection, Immunofluorescence, Expressing, Staining, Activity Assay, MTT Assay

    In vitro proof of rASC apoptosis at 24 h after incubation with Mitomycin (MMC) (a) Confocal microscopy of viable and MMC treated, apoptotic rASCs demonstrates cleaved Caspase-3 (green fluorescent indicates activated caspase-3; red fluorescent shows F-actin filaments; and DAPI displays cell nucleus with blue fluorescent). (b) Flow cytometry (Ex/Em 488/530) analysis of viable (left panel) and apoptotic rASCs (right panel) which were stained with FLICA kit (fluorescent inhibitor of caspases). (c) Quantification of cleaved caspase-3 in viable and apoptotic rASCs by the SensoLyte Homogenous AMC caspase-3/7 kit. Upon caspase-3/7 cleavage, Ac-DEVD-AMC generates the AMC fluorophore which has bright blue fluorescence and can be quantified at Ex/Em=354 nm/442 nm. Data are displayed as means of three experiments in each group with standard errors. *** indicates significant differences between viable and apoptotic cells ( P

    Journal: ACS nano

    Article Title: MR imaging of stem cell apoptosis in arthritic joints with a caspase-activatable contrast agent

    doi: 10.1021/nn504494c

    Figure Lengend Snippet: In vitro proof of rASC apoptosis at 24 h after incubation with Mitomycin (MMC) (a) Confocal microscopy of viable and MMC treated, apoptotic rASCs demonstrates cleaved Caspase-3 (green fluorescent indicates activated caspase-3; red fluorescent shows F-actin filaments; and DAPI displays cell nucleus with blue fluorescent). (b) Flow cytometry (Ex/Em 488/530) analysis of viable (left panel) and apoptotic rASCs (right panel) which were stained with FLICA kit (fluorescent inhibitor of caspases). (c) Quantification of cleaved caspase-3 in viable and apoptotic rASCs by the SensoLyte Homogenous AMC caspase-3/7 kit. Upon caspase-3/7 cleavage, Ac-DEVD-AMC generates the AMC fluorophore which has bright blue fluorescence and can be quantified at Ex/Em=354 nm/442 nm. Data are displayed as means of three experiments in each group with standard errors. *** indicates significant differences between viable and apoptotic cells ( P

    Article Snippet: To determine whether C-SNAM is a substrate for cleaved caspase-3, C-SNAM (200 μM) was incubated with human recombinant caspase-3 (50 nM, R & D systems), and the reaction was monitored by using HPLC and by measuring the molecular weight with HRMS.

    Techniques: In Vitro, Incubation, Confocal Microscopy, Flow Cytometry, Cytometry, Staining, Fluorescence

    Corresponding histopathology of viable and apoptotic rASC implants (a) H  E stains show MASI implant in the knee joint (b) DAPI stain shows cell nucleus (blue fluorescent), (c) red fluorescent demonstrates cleaved caspase 3 in apoptotic rASCs, (d) Overlay of b and c. (Scale bar=100 μm).

    Journal: ACS nano

    Article Title: MR imaging of stem cell apoptosis in arthritic joints with a caspase-activatable contrast agent

    doi: 10.1021/nn504494c

    Figure Lengend Snippet: Corresponding histopathology of viable and apoptotic rASC implants (a) H E stains show MASI implant in the knee joint (b) DAPI stain shows cell nucleus (blue fluorescent), (c) red fluorescent demonstrates cleaved caspase 3 in apoptotic rASCs, (d) Overlay of b and c. (Scale bar=100 μm).

    Article Snippet: To determine whether C-SNAM is a substrate for cleaved caspase-3, C-SNAM (200 μM) was incubated with human recombinant caspase-3 (50 nM, R & D systems), and the reaction was monitored by using HPLC and by measuring the molecular weight with HRMS.

    Techniques: Histopathology, Staining

    In vitro characterization of C-SNAM (a) HPLC traces of C-SNAM (black) and the incubation of C-SNAM (200 μM) with recombinant human caspase-3 (50 nM) in the caspase buffer at 37 °C overnight (red). (b) TEM images of GdNPs formed in the solution of C-SNAM (200 μM) following incubation with caspase-3 (50 nM) in caspase buffer (pH 7.4) overnight. Scale bar: 500 nm. (c) T1-weighted images show brighter MR signal for C-SNAM than other probes upon caspase-3 incubation. C-SNAM and Gd-DOTA (Dotarem®) at 200 μM in enzyme reaction buffer were incubated with and without caspase-3 (50 nM) at 37 °C overnight. T1-weighted FLASH images (TR/TE=161/6 ms) of the incubation solutions were acquired at 1T at 37 °C.

    Journal: ACS nano

    Article Title: MR imaging of stem cell apoptosis in arthritic joints with a caspase-activatable contrast agent

    doi: 10.1021/nn504494c

    Figure Lengend Snippet: In vitro characterization of C-SNAM (a) HPLC traces of C-SNAM (black) and the incubation of C-SNAM (200 μM) with recombinant human caspase-3 (50 nM) in the caspase buffer at 37 °C overnight (red). (b) TEM images of GdNPs formed in the solution of C-SNAM (200 μM) following incubation with caspase-3 (50 nM) in caspase buffer (pH 7.4) overnight. Scale bar: 500 nm. (c) T1-weighted images show brighter MR signal for C-SNAM than other probes upon caspase-3 incubation. C-SNAM and Gd-DOTA (Dotarem®) at 200 μM in enzyme reaction buffer were incubated with and without caspase-3 (50 nM) at 37 °C overnight. T1-weighted FLASH images (TR/TE=161/6 ms) of the incubation solutions were acquired at 1T at 37 °C.

    Article Snippet: To determine whether C-SNAM is a substrate for cleaved caspase-3, C-SNAM (200 μM) was incubated with human recombinant caspase-3 (50 nM, R & D systems), and the reaction was monitored by using HPLC and by measuring the molecular weight with HRMS.

    Techniques: In Vitro, High Performance Liquid Chromatography, Incubation, Recombinant, Transmission Electron Microscopy, Mass Spectrometry

    General design and mechanism of action of the caspase-3 sensitive nano-aggregation MRI probe (C-SNAM) (a) Chemical structure of C-SNAM ( 1 ). Following disulfide reduction and caspase-3-triggered DEVD peptide cleavage, C-SNAM transforms to a rigid and hydrophobic cyclized product 2 , through a biocompatible intramolecular cyclization reaction between 2-cyano-6-hydroxyquinoline (CHQ) and D -cysteine residue. The macrocycle 2 will subsequently self-assemble into Gd-nanoparticles (GdNPs) due to the increased intermolecular interactions (i.e. hydrophobic interactions, π-π stacking), leading to an increase in r 1 relaxivity relative to the unactivated probe 1. (b) Corresponding mechanism of action in vivo. (1) Intra-articular injection of C-SNAM into rat knee joints with implants of apoptotic and viable stem cells. (2) In vivo activation of C-SNAM in apoptotic stem cell transplants through caspase-3 mediated activation. (3) Increased relaxivity and retention effect of GdNPs leads to enhanced MRI signal of apoptotic stem cell transplants.

    Journal: ACS nano

    Article Title: MR imaging of stem cell apoptosis in arthritic joints with a caspase-activatable contrast agent

    doi: 10.1021/nn504494c

    Figure Lengend Snippet: General design and mechanism of action of the caspase-3 sensitive nano-aggregation MRI probe (C-SNAM) (a) Chemical structure of C-SNAM ( 1 ). Following disulfide reduction and caspase-3-triggered DEVD peptide cleavage, C-SNAM transforms to a rigid and hydrophobic cyclized product 2 , through a biocompatible intramolecular cyclization reaction between 2-cyano-6-hydroxyquinoline (CHQ) and D -cysteine residue. The macrocycle 2 will subsequently self-assemble into Gd-nanoparticles (GdNPs) due to the increased intermolecular interactions (i.e. hydrophobic interactions, π-π stacking), leading to an increase in r 1 relaxivity relative to the unactivated probe 1. (b) Corresponding mechanism of action in vivo. (1) Intra-articular injection of C-SNAM into rat knee joints with implants of apoptotic and viable stem cells. (2) In vivo activation of C-SNAM in apoptotic stem cell transplants through caspase-3 mediated activation. (3) Increased relaxivity and retention effect of GdNPs leads to enhanced MRI signal of apoptotic stem cell transplants.

    Article Snippet: To determine whether C-SNAM is a substrate for cleaved caspase-3, C-SNAM (200 μM) was incubated with human recombinant caspase-3 (50 nM, R & D systems), and the reaction was monitored by using HPLC and by measuring the molecular weight with HRMS.

    Techniques: Magnetic Resonance Imaging, In Vivo, Injection, Activation Assay

    Immunohistochemistry and immunofluorescence staining of stem cell apoptosis and macrophage infiltration in calvarial defects of mice transplanted with Feru-AFC NP-labeled pMSCs (left panel) and mMSCs (right panel). (a, h) Hematoxylin and eosin (H E) staining of the tissue sections shows the scaffold embedded with pMSCs or mMSCs above the brain. (b, i) Prussian blue staining shows the presence of Feru-AFC NPs within the scaffold (black arrowheads). (c, j) Higher levels of activated caspase-3 (brown stain) were detected in pMSCs compared to mMSCs (black arrowheads). (d, k) Immunofluorescence staining shows higher levels of activated caspase-3 (red) in pMSCs compared to mMSCs (white arrowheads). DAPI was used to counterstain for the nucleus (blue). (e, l) Higher amounts of mouse macrophage (CD68, brown) infiltration was detected in the scaffold embedded with pMSCs (black arrowheads) versus mMSCs. (f, m) Immunofluorescence staining shows higher amounts of mouse macrophage (CD68, red) infiltration for in the scaffold embedded with pMSCs (white arrows) versus mMSCs. Feru-AFC NPs were detected by staining for dextran (green). DAPI was used to counterstain for the nucleus (blue). (g, n) H E staining (4× stitched images) of the scaffold seeded with labeled pMSCs (left panel) and mMSCs (right panel). The scale bar in each panel represents 50 µm.

    Journal: Nanotheranostics

    Article Title: Ferumoxytol-based Dual-modality Imaging Probe for Detection of Stem Cell Transplant Rejection

    doi: 10.7150/ntno.26389

    Figure Lengend Snippet: Immunohistochemistry and immunofluorescence staining of stem cell apoptosis and macrophage infiltration in calvarial defects of mice transplanted with Feru-AFC NP-labeled pMSCs (left panel) and mMSCs (right panel). (a, h) Hematoxylin and eosin (H E) staining of the tissue sections shows the scaffold embedded with pMSCs or mMSCs above the brain. (b, i) Prussian blue staining shows the presence of Feru-AFC NPs within the scaffold (black arrowheads). (c, j) Higher levels of activated caspase-3 (brown stain) were detected in pMSCs compared to mMSCs (black arrowheads). (d, k) Immunofluorescence staining shows higher levels of activated caspase-3 (red) in pMSCs compared to mMSCs (white arrowheads). DAPI was used to counterstain for the nucleus (blue). (e, l) Higher amounts of mouse macrophage (CD68, brown) infiltration was detected in the scaffold embedded with pMSCs (black arrowheads) versus mMSCs. (f, m) Immunofluorescence staining shows higher amounts of mouse macrophage (CD68, red) infiltration for in the scaffold embedded with pMSCs (white arrows) versus mMSCs. Feru-AFC NPs were detected by staining for dextran (green). DAPI was used to counterstain for the nucleus (blue). (g, n) H E staining (4× stitched images) of the scaffold seeded with labeled pMSCs (left panel) and mMSCs (right panel). The scale bar in each panel represents 50 µm.

    Article Snippet: No recombinant human caspase-3 was added in the control group.

    Techniques: Immunohistochemistry, Immunofluorescence, Staining, Mouse Assay, Labeling

    Design and mechanism of a caspase-3-cleavable probe with fluorescence “light-up” signature based on ferumoxytol NPs. (a) The chemical structure of the activated caspase-3-cleavable peptide, KKKKDEVD-AFC, and the schematic illustration of the fluorescence response of Feru-AFC NPs to activated caspase-3. The Feru-AFC NPs remain in an “off” state when the peptide is intact. In the presence of activated caspase-3, DEVD is cleaved to release AFC molecules (“on” state) from ferumoxytol with intense fluorescence that can be detected in a FITC channel at an emission maximum at 495 nm. (b) Schematic illustration of the in vivo evaluation of activated caspase-3-cleavable Feru-AFC NPs. The recipient mice received calvarial defects, followed by transplantation of mMSCs (matched) or pMSCs (mismatched) labeled with Feru-AFC NPs and seeded in polyethylene glycol (PEG)-based scaffolds. Apoptosis of mismatched pMSCs by immune rejection activates the cleavage of the peptide for increased fluorescence signal upon laser excitation.

    Journal: Nanotheranostics

    Article Title: Ferumoxytol-based Dual-modality Imaging Probe for Detection of Stem Cell Transplant Rejection

    doi: 10.7150/ntno.26389

    Figure Lengend Snippet: Design and mechanism of a caspase-3-cleavable probe with fluorescence “light-up” signature based on ferumoxytol NPs. (a) The chemical structure of the activated caspase-3-cleavable peptide, KKKKDEVD-AFC, and the schematic illustration of the fluorescence response of Feru-AFC NPs to activated caspase-3. The Feru-AFC NPs remain in an “off” state when the peptide is intact. In the presence of activated caspase-3, DEVD is cleaved to release AFC molecules (“on” state) from ferumoxytol with intense fluorescence that can be detected in a FITC channel at an emission maximum at 495 nm. (b) Schematic illustration of the in vivo evaluation of activated caspase-3-cleavable Feru-AFC NPs. The recipient mice received calvarial defects, followed by transplantation of mMSCs (matched) or pMSCs (mismatched) labeled with Feru-AFC NPs and seeded in polyethylene glycol (PEG)-based scaffolds. Apoptosis of mismatched pMSCs by immune rejection activates the cleavage of the peptide for increased fluorescence signal upon laser excitation.

    Article Snippet: No recombinant human caspase-3 was added in the control group.

    Techniques: Fluorescence, In Vivo, Mouse Assay, Transplantation Assay, Labeling

    Correlation of LDH to caspase 3/7 Activity in Nasal Wash.

    Journal: Influenza and Other Respiratory Viruses

    Article Title: Lactate dehydrogenase and caspase activity in nasopharyngeal secretions are predictors of bronchiolitis severity

    doi: 10.1111/irv.12276

    Figure Lengend Snippet: Correlation of LDH to caspase 3/7 Activity in Nasal Wash.

    Article Snippet: To calculate relative units (U/l) of caspase activity, a standard curve using recombinant human caspase-3 protein (R & D Systems, Minneapolis, MN, USA) was used demonstrating an ample linear dynamic range of activity at the dilutions tested (20 000–32 U/l).

    Techniques: Activity Assay

    (A) LDH in Nasal Wash ((log 10  mU/l) Compared to Different Viruses. (B) Caspase 3/7 Activity in Nasal Wash compared to Different Viruses.

    Journal: Influenza and Other Respiratory Viruses

    Article Title: Lactate dehydrogenase and caspase activity in nasopharyngeal secretions are predictors of bronchiolitis severity

    doi: 10.1111/irv.12276

    Figure Lengend Snippet: (A) LDH in Nasal Wash ((log 10 mU/l) Compared to Different Viruses. (B) Caspase 3/7 Activity in Nasal Wash compared to Different Viruses.

    Article Snippet: To calculate relative units (U/l) of caspase activity, a standard curve using recombinant human caspase-3 protein (R & D Systems, Minneapolis, MN, USA) was used demonstrating an ample linear dynamic range of activity at the dilutions tested (20 000–32 U/l).

    Techniques: Activity Assay