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    Structured Review

    Millipore caspase assay buffer
    Protein and mRNA analysis of the inflammasome components in the heart of wild-type mice with or without LPS low dose. ( A ) NLRP3, <t>pro-caspase-1,</t> and ASC protein expression were analysed by western blot and ( B ) were quantified through densitometric analysis.
    Caspase Assay Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Independent roles of the priming and the triggering of the NLRP3 inflammasome in the heart"

    Article Title: Independent roles of the priming and the triggering of the NLRP3 inflammasome in the heart

    Journal: Cardiovascular Research

    doi: 10.1093/cvr/cvu259

    Protein and mRNA analysis of the inflammasome components in the heart of wild-type mice with or without LPS low dose. ( A ) NLRP3, pro-caspase-1, and ASC protein expression were analysed by western blot and ( B ) were quantified through densitometric analysis.
    Figure Legend Snippet: Protein and mRNA analysis of the inflammasome components in the heart of wild-type mice with or without LPS low dose. ( A ) NLRP3, pro-caspase-1, and ASC protein expression were analysed by western blot and ( B ) were quantified through densitometric analysis.

    Techniques Used: Mouse Assay, Expressing, Western Blot

    Catalytic activity and mRNA quantification of caspase-1 in spleen and heart of wt and Nlrp3-A350V/CreT mice. ( A ) Enzymatic activity of caspase-1 reported as relative fluorescence produced compared with controls ( n = 5 per group). Values are expressed
    Figure Legend Snippet: Catalytic activity and mRNA quantification of caspase-1 in spleen and heart of wt and Nlrp3-A350V/CreT mice. ( A ) Enzymatic activity of caspase-1 reported as relative fluorescence produced compared with controls ( n = 5 per group). Values are expressed

    Techniques Used: Activity Assay, Mouse Assay, Fluorescence, Produced

    Effects of low-dose LPS in wild-type, Nlrp3-A350V , and KI Nlrp3-A350V/CreT mice. ( A ) Caspase-1 activity in the heart of wild type, Nlrp3-A350V , and Nlrp3-A350V/CreT treated with tamoxifen and then challenged with low-dose LPS or the vehicle solution for
    Figure Legend Snippet: Effects of low-dose LPS in wild-type, Nlrp3-A350V , and KI Nlrp3-A350V/CreT mice. ( A ) Caspase-1 activity in the heart of wild type, Nlrp3-A350V , and Nlrp3-A350V/CreT treated with tamoxifen and then challenged with low-dose LPS or the vehicle solution for

    Techniques Used: Mouse Assay, Activity Assay

    2) Product Images from "Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against osteosarcoma"

    Article Title: Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against osteosarcoma

    Journal: Cancer Biology & Therapy

    doi: 10.1080/15384047.2015.1017155

    NVP-BEZ235 activates apoptosis in osteosarcoma cells . MG-63 cells were treated with indicated concentration of NVP-BEZ235 (NVP) for 42 hrs, cell apoptosis was tested by caspase-3 activity assay ( A ), Histone-DNA ELISA assay ( B ) and TUNEL staining assay ( C ). The expression of caspase-3 (regular and cleaved) and tubulin (equal loading) was tested by Western blotting ( A , upper panel). MG-63 cells were pretreated with z-VAD-fmk (zvad, 25 μM), z-DVED-fmk (dved, 25 μM) or z-ITED-fmk (ited, 25 μM) for 1 hr, followed by NVP-BEZ235 (200 nM) stimulation, cells were further cultured, before cell growth ( D ) and colony formation ( E ) were tested. Apoptosis in NVP-BEZ235 (200 nM, 42 hrs)-treated U2OS and SaOs-2 cells was analyzed by TUNEL staining assay ( F ). n = 5 for each assay. * P
    Figure Legend Snippet: NVP-BEZ235 activates apoptosis in osteosarcoma cells . MG-63 cells were treated with indicated concentration of NVP-BEZ235 (NVP) for 42 hrs, cell apoptosis was tested by caspase-3 activity assay ( A ), Histone-DNA ELISA assay ( B ) and TUNEL staining assay ( C ). The expression of caspase-3 (regular and cleaved) and tubulin (equal loading) was tested by Western blotting ( A , upper panel). MG-63 cells were pretreated with z-VAD-fmk (zvad, 25 μM), z-DVED-fmk (dved, 25 μM) or z-ITED-fmk (ited, 25 μM) for 1 hr, followed by NVP-BEZ235 (200 nM) stimulation, cells were further cultured, before cell growth ( D ) and colony formation ( E ) were tested. Apoptosis in NVP-BEZ235 (200 nM, 42 hrs)-treated U2OS and SaOs-2 cells was analyzed by TUNEL staining assay ( F ). n = 5 for each assay. * P

    Techniques Used: Concentration Assay, Caspase-3 Activity Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Expressing, Western Blot, Cell Culture

    3) Product Images from "Cordycepin Down-Regulates Multiple Drug Resistant (MDR)/HIF-1α through Regulating AMPK/mTORC1 Signaling in GBC-SD Gallbladder Cancer Cells"

    Article Title: Cordycepin Down-Regulates Multiple Drug Resistant (MDR)/HIF-1α through Regulating AMPK/mTORC1 Signaling in GBC-SD Gallbladder Cancer Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms150712778

    Cordycepin induces GBC-SD cell apoptosis. GBC-SD cells were treated with indicated cordycepin (10–100 μM) for 72 h, cell apoptosis was tested by Annexin V/PI FACS ( A – C ) and Caspase-3 activity assay ( D ); Note that for Annexin V FACS assay, the results of five sets of experiments were quantified ( B , C ); GBC-SD cells were treated with z-VAD-fmk (50 μM) for 1 h, followed by cordycepin (25/100 μM) stimulation, cells were further cultured for 72 h, and cell viability was tested ( E ); Experiments were repeated five times. * Stands for p
    Figure Legend Snippet: Cordycepin induces GBC-SD cell apoptosis. GBC-SD cells were treated with indicated cordycepin (10–100 μM) for 72 h, cell apoptosis was tested by Annexin V/PI FACS ( A – C ) and Caspase-3 activity assay ( D ); Note that for Annexin V FACS assay, the results of five sets of experiments were quantified ( B , C ); GBC-SD cells were treated with z-VAD-fmk (50 μM) for 1 h, followed by cordycepin (25/100 μM) stimulation, cells were further cultured for 72 h, and cell viability was tested ( E ); Experiments were repeated five times. * Stands for p

    Techniques Used: FACS, Caspase-3 Activity Assay, Cell Culture

    4) Product Images from "Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings"

    Article Title: Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9969

    ODE activates apoptosis in CRC cells A panel of established CRC cell lines (HCT-116, Lovo, HT-29 and DLD-1) and three primary human CRC cell lines were treated with or without ODE at applied concentrations, cells were further cultured, cell apoptosis was analyzed by listed assay A - D , G and H . HCT-116 cells were pretreated with Ac-DEVD-CHO (“DVED”), Ac-LEHD-CHO (“LEHD”) or Ac-VAD-CHO (“VAD”) (40 μM each) for 1 h, following by ODE (50 μg/mL) treatment, cell viability E . and cell death F . were tested. “DMSO” stands for 0.1% DMSO. Cleaved-PARP/cleaved-caspase-3 expression (vs. Tubulin) was quantified. Data in this figure were repeated four times, and similar results were obtained. * p
    Figure Legend Snippet: ODE activates apoptosis in CRC cells A panel of established CRC cell lines (HCT-116, Lovo, HT-29 and DLD-1) and three primary human CRC cell lines were treated with or without ODE at applied concentrations, cells were further cultured, cell apoptosis was analyzed by listed assay A - D , G and H . HCT-116 cells were pretreated with Ac-DEVD-CHO (“DVED”), Ac-LEHD-CHO (“LEHD”) or Ac-VAD-CHO (“VAD”) (40 μM each) for 1 h, following by ODE (50 μg/mL) treatment, cell viability E . and cell death F . were tested. “DMSO” stands for 0.1% DMSO. Cleaved-PARP/cleaved-caspase-3 expression (vs. Tubulin) was quantified. Data in this figure were repeated four times, and similar results were obtained. * p

    Techniques Used: Cell Culture, Expressing

    5) Product Images from "The anti-cancer activity of the mTORC1/2 dual inhibitor XL388 in preclinical osteosarcoma models"

    Article Title: The anti-cancer activity of the mTORC1/2 dual inhibitor XL388 in preclinical osteosarcoma models

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10389

    XL388 induces caspase-dependent apoptosis activation in OS cells-MG-63 ( A–C ), U2OS ( F ) and SaOs-2 (F) OS cell lines, the primary human OS cells (line-1/−3) ( G and H ), as well as mouse osteoblastic MC3T3-E1 cells (F) and human OB-6 osteoblastic cells (G and H) were treated with applied concentration of XL388, cells were further cultured, and cell apoptosis was tested by listed assays. MG-63 cells, pretreated with z-VAD-fmk (vad, 25 μM), z-DVED-fmk (dved, 25 μM) or z-ITED-fmk (ited, 25 μM) for 1 hour, were stimulated with XL388 (100 nM), TUNEL staining assay ( D ) and colony formation assay ( E ) were performed. n = 5 for each assay. * p
    Figure Legend Snippet: XL388 induces caspase-dependent apoptosis activation in OS cells-MG-63 ( A–C ), U2OS ( F ) and SaOs-2 (F) OS cell lines, the primary human OS cells (line-1/−3) ( G and H ), as well as mouse osteoblastic MC3T3-E1 cells (F) and human OB-6 osteoblastic cells (G and H) were treated with applied concentration of XL388, cells were further cultured, and cell apoptosis was tested by listed assays. MG-63 cells, pretreated with z-VAD-fmk (vad, 25 μM), z-DVED-fmk (dved, 25 μM) or z-ITED-fmk (ited, 25 μM) for 1 hour, were stimulated with XL388 (100 nM), TUNEL staining assay ( D ) and colony formation assay ( E ) were performed. n = 5 for each assay. * p

    Techniques Used: Activation Assay, Concentration Assay, Cell Culture, TUNEL Assay, Staining, Colony Assay

    6) Product Images from "Selective mGluR1 Antagonist EMQMCM Inhibits the Kainate-Induced Excitotoxicity in Primary Neuronal Cultures and in the Rat Hippocampus"

    Article Title: Selective mGluR1 Antagonist EMQMCM Inhibits the Kainate-Induced Excitotoxicity in Primary Neuronal Cultures and in the Rat Hippocampus

    Journal: Neurotoxicity Research

    doi: 10.1007/s12640-011-9293-4

    a , c The effect of EMQMCM on kainate (150 μM)-induced LDH release in the primary cultures of mouse cortical ( a ) and hippocampal ( c ) neurons. LDH was measured 48 h (cortical) or 24 h (hippocampal cultures) after KA administration. EMQMCM (in concentrations 0.1, 1, 10, and 100 μM) was added to the culture medium 30 min, 1 h, 3 h, or 6 h after KA. b , d The effect of EMQMCM on KA-induced increase in caspase-3 activity in mouse primary cortical ( b ) and hippocampal cultures ( d ). Caspase-3 was measured 6 h after starting KA intoxication. EMQMCM was added to cultures 30 min after KA. Each bar represents the mean of n ≥ 6 platings ± SEM from 3 to 4 independent experiments. Significant differences marked in the following way: *** P
    Figure Legend Snippet: a , c The effect of EMQMCM on kainate (150 μM)-induced LDH release in the primary cultures of mouse cortical ( a ) and hippocampal ( c ) neurons. LDH was measured 48 h (cortical) or 24 h (hippocampal cultures) after KA administration. EMQMCM (in concentrations 0.1, 1, 10, and 100 μM) was added to the culture medium 30 min, 1 h, 3 h, or 6 h after KA. b , d The effect of EMQMCM on KA-induced increase in caspase-3 activity in mouse primary cortical ( b ) and hippocampal cultures ( d ). Caspase-3 was measured 6 h after starting KA intoxication. EMQMCM was added to cultures 30 min after KA. Each bar represents the mean of n ≥ 6 platings ± SEM from 3 to 4 independent experiments. Significant differences marked in the following way: *** P

    Techniques Used: Activity Assay

    7) Product Images from "Effect of Chemotherapeutic Drugs on Caspase-3 Activity, as a Key Biomarker for Apoptosis in Ovarian Tumor Cell Cultured as Monolayer. A Pilot Study"

    Article Title: Effect of Chemotherapeutic Drugs on Caspase-3 Activity, as a Key Biomarker for Apoptosis in Ovarian Tumor Cell Cultured as Monolayer. A Pilot Study

    Journal: Iranian Journal of Pharmaceutical Research : IJPR

    doi:

    Effect of combined chemotherapeutic drugs on tumor cell apoptosis. Cells were treated with paclitaxel plus doxorubicin (P + D), paclitaxel plus carboplatin (P + C), paclitaxel plus endoxan (P + E), doxorubicin plus carboplatin (D + C), doxorubicin plus endoxan (D + E), or carboplatin plus endoxan (C + E) for 6 days. Apoptosis was measured using a caspase-3 assay. *p
    Figure Legend Snippet: Effect of combined chemotherapeutic drugs on tumor cell apoptosis. Cells were treated with paclitaxel plus doxorubicin (P + D), paclitaxel plus carboplatin (P + C), paclitaxel plus endoxan (P + E), doxorubicin plus carboplatin (D + C), doxorubicin plus endoxan (D + E), or carboplatin plus endoxan (C + E) for 6 days. Apoptosis was measured using a caspase-3 assay. *p

    Techniques Used: Caspase-3 Assay

    Effect of individual chemotherapeutic drugs on tumor cell apoptosis. Cells were treated with paclitaxel (13.8µM), doxorubicin (2.5µM), carboplatin (13.4µM) or endoxan (19µM) for 6 days. Apoptosis was measured using a caspase-3 assay. *p
    Figure Legend Snippet: Effect of individual chemotherapeutic drugs on tumor cell apoptosis. Cells were treated with paclitaxel (13.8µM), doxorubicin (2.5µM), carboplatin (13.4µM) or endoxan (19µM) for 6 days. Apoptosis was measured using a caspase-3 assay. *p

    Techniques Used: Caspase-3 Assay

    8) Product Images from "The preclinical assessment of XL388, a mTOR kinase inhibitor, as a promising anti-renal cell carcinoma agent"

    Article Title: The preclinical assessment of XL388, a mTOR kinase inhibitor, as a promising anti-renal cell carcinoma agent

    Journal: Oncotarget

    doi: 10.18632/oncotarget.15620

    XL388 activates apoptosis in RCC cells 786-0 or A498 RCC cells, the primary human RCC cells (“RCC1 and RCC2”) or the HK-2 cells were stimulated with applied concentration of XL388, cells were further cultured in the conditional medium for applied time, cell apoptosis was tested by the caspase activity assay A ., TUNEL staining assay B and F . and the ssDNA ELISA assay C . 786-0 cells were pre-treated for 30 min with 50 μM of the caspase-9 inhibitor z-LEHD-CHO (“+lehd”), the caspase-3 inhibitor z-DEVD-CHO (“+devd”) or the pan caspase inhibitor z-VAD-CHO (“+vad”), followed by XL388 (500 nM) treatment, cell apoptosis and viability were tested by the TUNEL assay D . and the CCK-8 assay E ., respectively. For each assay, n=5. Experiments in this figure were repeated three times, and similar results were obtained. * p
    Figure Legend Snippet: XL388 activates apoptosis in RCC cells 786-0 or A498 RCC cells, the primary human RCC cells (“RCC1 and RCC2”) or the HK-2 cells were stimulated with applied concentration of XL388, cells were further cultured in the conditional medium for applied time, cell apoptosis was tested by the caspase activity assay A ., TUNEL staining assay B and F . and the ssDNA ELISA assay C . 786-0 cells were pre-treated for 30 min with 50 μM of the caspase-9 inhibitor z-LEHD-CHO (“+lehd”), the caspase-3 inhibitor z-DEVD-CHO (“+devd”) or the pan caspase inhibitor z-VAD-CHO (“+vad”), followed by XL388 (500 nM) treatment, cell apoptosis and viability were tested by the TUNEL assay D . and the CCK-8 assay E ., respectively. For each assay, n=5. Experiments in this figure were repeated three times, and similar results were obtained. * p

    Techniques Used: Concentration Assay, Cell Culture, Caspase Activity Assay, TUNEL Assay, Staining, Enzyme-linked Immunosorbent Assay, CCK-8 Assay

    9) Product Images from "The Antipancreatic Cancer Activity of OSI-027, a Potent and Selective Inhibitor of mTORC1 and mTORC2"

    Article Title: The Antipancreatic Cancer Activity of OSI-027, a Potent and Selective Inhibitor of mTORC1 and mTORC2

    Journal: DNA and Cell Biology

    doi: 10.1089/dna.2015.2886

    OSI-027 induces human pancreatic cancer cell apoptosis in vitro . Human pancreatic cancer lines (PANC-1 or MIA PaCa-2) were either left untreated (“C”), or stimulated with applied concentration of OSI-027, cells were then cultured for an indicated time, and cell apoptosis was analyzed by staining assay (A, B) and caspase-3 activity assay (C, D) . PANC-1 or primary human pancreatic cancer cells, pretreated with the caspase-3 inhibitor z-DVED-fmk (ZDVED, 30 μM), the caspase-8 inhibitor z-ITED-fmk (ITED, 30 μM), or the pan caspase inhibitor z-VAD-fmk (ZVAD, 30 μM) for 1 h, were stimulated with OSI-027 (100 nM) for 72 h, cell survival was analyzed by CellTiter-Glo assay (E, F) . Data are expressed as mean±SE, experiments were repeated five times. n =5 for each assay. * p
    Figure Legend Snippet: OSI-027 induces human pancreatic cancer cell apoptosis in vitro . Human pancreatic cancer lines (PANC-1 or MIA PaCa-2) were either left untreated (“C”), or stimulated with applied concentration of OSI-027, cells were then cultured for an indicated time, and cell apoptosis was analyzed by staining assay (A, B) and caspase-3 activity assay (C, D) . PANC-1 or primary human pancreatic cancer cells, pretreated with the caspase-3 inhibitor z-DVED-fmk (ZDVED, 30 μM), the caspase-8 inhibitor z-ITED-fmk (ITED, 30 μM), or the pan caspase inhibitor z-VAD-fmk (ZVAD, 30 μM) for 1 h, were stimulated with OSI-027 (100 nM) for 72 h, cell survival was analyzed by CellTiter-Glo assay (E, F) . Data are expressed as mean±SE, experiments were repeated five times. n =5 for each assay. * p

    Techniques Used: In Vitro, Concentration Assay, Cell Culture, Staining, Caspase-3 Activity Assay, Glo Assay

    10) Product Images from "ROS-p53-cyclophilin-D signaling mediates salinomycin-induced glioma cell necrosis"

    Article Title: ROS-p53-cyclophilin-D signaling mediates salinomycin-induced glioma cell necrosis

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-015-0174-1

    Salinomycin induces both apoptotic and necrotic death of glioma cells. U87MG cells were treated with indicated concentration of salinomycin (Sali) for 72 h, cell apoptosis was analyzed by Annexin V FACS assay ( a ) and caspase-3 activity assay ( b ). The effect of zVADfmk (60 μM) on salinomycin (Sali, 5 μM)-induced apoptosis was also shown ( a - b ). The expression of cleaved-caspase-3, cytochrome C and tubulin in cytosol after indicated salinomycin (Sali) stimulation was tested by Western blotting ( c ). U87MG cells were pre-treated with an apoptosis inhibitor zVADfmk (30 or 60 μM) for 1 h, followed by salinomycin (Sali, 5 μM) stimulation, cells were further cultured, MTT assay ( d , 72 h) and trypan blue staining ( e , 72 h) were performed. U251MG and EFC-2 glioma cells were treated with salinomycin (Sali, 5 μM) in the presence or absence of zVADfmk (zVAD, 60 μM) for 72 h, cell death was tested by trypan blue staining assay ( f ). U87MG cells were pre-treated with a necrosis inhibitor Nec-1 (25 μM) for 1 h, followed by salinomycin (Sali, 5/10 μM) stimulation, cells were further cultured for 72 h, cell necrosis was analyzed by FACS ( g ). The cell viability of U87MG cells with indicated treatment was tested ( h ). Experiments were repeated four times in this figure, and similar results were obtained. Error bars indicate SD. * p
    Figure Legend Snippet: Salinomycin induces both apoptotic and necrotic death of glioma cells. U87MG cells were treated with indicated concentration of salinomycin (Sali) for 72 h, cell apoptosis was analyzed by Annexin V FACS assay ( a ) and caspase-3 activity assay ( b ). The effect of zVADfmk (60 μM) on salinomycin (Sali, 5 μM)-induced apoptosis was also shown ( a - b ). The expression of cleaved-caspase-3, cytochrome C and tubulin in cytosol after indicated salinomycin (Sali) stimulation was tested by Western blotting ( c ). U87MG cells were pre-treated with an apoptosis inhibitor zVADfmk (30 or 60 μM) for 1 h, followed by salinomycin (Sali, 5 μM) stimulation, cells were further cultured, MTT assay ( d , 72 h) and trypan blue staining ( e , 72 h) were performed. U251MG and EFC-2 glioma cells were treated with salinomycin (Sali, 5 μM) in the presence or absence of zVADfmk (zVAD, 60 μM) for 72 h, cell death was tested by trypan blue staining assay ( f ). U87MG cells were pre-treated with a necrosis inhibitor Nec-1 (25 μM) for 1 h, followed by salinomycin (Sali, 5/10 μM) stimulation, cells were further cultured for 72 h, cell necrosis was analyzed by FACS ( g ). The cell viability of U87MG cells with indicated treatment was tested ( h ). Experiments were repeated four times in this figure, and similar results were obtained. Error bars indicate SD. * p

    Techniques Used: Concentration Assay, FACS, Caspase-3 Activity Assay, Expressing, Western Blot, Cell Culture, MTT Assay, Staining

    11) Product Images from "Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity"

    Article Title: Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20172222

    Caspase-1 processing at the CDL destabilizes caspase-1 dimers and terminates protease activity. Bone marrow progenitors from Ice −/− ( Casp1 −/− /Casp11 null/null ) mice were retrovirally reconstituted for mouse caspase-1 (WT, C284 catalytic mutant, or CDL-uncleavable mutant) during their differentiation to macrophages. (A) Differentiated macrophages were then left untreated or LPS-treated for 6 h, or were LPS-primed for 4 h before stimulation with nigericin for 0.5, 1, or 2 h. Cell extracts and cell-free supernatants were mixed and analyzed by immunoblot. (B) Differentiated, transduced macrophages were LPS-primed for 4 h, before stimulation with nigericin for 30 min. Cell culture medium was then replaced with caspase assay buffer containing the caspase-1 fluorogenic substrate, YVAD-afc, and caspase-1–like activity was monitored over the next 30 min. (C) An engineered caspase-1 system allowed controlled caspase-1 dimerization by AP20187 (AP) and CDL cleavage by thrombin (thr). 1 µM AP20187 was added to HEK293T-expressing caspase-1 constructs (WT vs. catalytic mutant, C284A) for 10 min. Cell culture medium was replaced for caspase activity buffer supplemented with digitonin to lyse cells for activity assays (± 20 U/ml thrombin). (D) Immunoblot analysis of caspase-1 cleavage by thrombin after incubation for 1 h. (E) Caspase activity assay, in which thrombin was added to cell lysates at the same time as the YVAD-afc fluorogenic substrate. (F) Cleavage assay in which thrombin was added to caspase-1–expressing cell lysates at the same time as natural substrates (lysates from HEK293T ectopically expressing V5-tagged substrates) and incubated for 0.5 h. (G) Thrombin was added to caspase-1_C284A–expressing cell lysates and incubated for 1 h. Bismaleimidohexane was then added to lysates for 1 h to cross-link caspase-1 dimers. Graphs are mean + SD of triplicate wells from a single experiment. All data are representative of at least two (G) or three (A–F) independent experiments.
    Figure Legend Snippet: Caspase-1 processing at the CDL destabilizes caspase-1 dimers and terminates protease activity. Bone marrow progenitors from Ice −/− ( Casp1 −/− /Casp11 null/null ) mice were retrovirally reconstituted for mouse caspase-1 (WT, C284 catalytic mutant, or CDL-uncleavable mutant) during their differentiation to macrophages. (A) Differentiated macrophages were then left untreated or LPS-treated for 6 h, or were LPS-primed for 4 h before stimulation with nigericin for 0.5, 1, or 2 h. Cell extracts and cell-free supernatants were mixed and analyzed by immunoblot. (B) Differentiated, transduced macrophages were LPS-primed for 4 h, before stimulation with nigericin for 30 min. Cell culture medium was then replaced with caspase assay buffer containing the caspase-1 fluorogenic substrate, YVAD-afc, and caspase-1–like activity was monitored over the next 30 min. (C) An engineered caspase-1 system allowed controlled caspase-1 dimerization by AP20187 (AP) and CDL cleavage by thrombin (thr). 1 µM AP20187 was added to HEK293T-expressing caspase-1 constructs (WT vs. catalytic mutant, C284A) for 10 min. Cell culture medium was replaced for caspase activity buffer supplemented with digitonin to lyse cells for activity assays (± 20 U/ml thrombin). (D) Immunoblot analysis of caspase-1 cleavage by thrombin after incubation for 1 h. (E) Caspase activity assay, in which thrombin was added to cell lysates at the same time as the YVAD-afc fluorogenic substrate. (F) Cleavage assay in which thrombin was added to caspase-1–expressing cell lysates at the same time as natural substrates (lysates from HEK293T ectopically expressing V5-tagged substrates) and incubated for 0.5 h. (G) Thrombin was added to caspase-1_C284A–expressing cell lysates and incubated for 1 h. Bismaleimidohexane was then added to lysates for 1 h to cross-link caspase-1 dimers. Graphs are mean + SD of triplicate wells from a single experiment. All data are representative of at least two (G) or three (A–F) independent experiments.

    Techniques Used: Activity Assay, Mouse Assay, Mutagenesis, Cell Culture, Caspase Assay, Expressing, Construct, Incubation, Caspase Activity Assay, Cleavage Assay

    Cell type specifies caspase-1 activity duration. (A–D) LPS-primed (A and B) macrophages or (A–D) neutrophils were left untreated or stimulated with nigericin. (A) ASC speck intensity of Ice −/− macrophages and neutrophils (4 h LPS + nigericin), quantified from microscopy images. Each circle is one speck ( n = 20–55), and significance between samples was assessed by nonparametric, unpaired Mann–Whitney test. (B) Immunoblot for inflammasome component expression in whole-cell extracts (4 h LPS). (C) Cell lysates and cell-free supernatants were harvested at various times after nigericin stimulation (0, 2, 4, 8 h), and analyzed by Western blot. *, Nonspecific band. (D) bVAD-fmk was applied at the indicated time points. Supernatant was removed, and cells were lysed 8 h after nigericin stimulation, and active caspase-1 was pulled down using streptavidin beads. Streptavidin-bound and -unbound fractions from mixed supernatants/extracts were analyzed by Western blot. All data are representative of at least three (A, B, C [WT], D [WT]) or two (C [ Ice −/− ], D [ Ice −/− ]) independent experiments.
    Figure Legend Snippet: Cell type specifies caspase-1 activity duration. (A–D) LPS-primed (A and B) macrophages or (A–D) neutrophils were left untreated or stimulated with nigericin. (A) ASC speck intensity of Ice −/− macrophages and neutrophils (4 h LPS + nigericin), quantified from microscopy images. Each circle is one speck ( n = 20–55), and significance between samples was assessed by nonparametric, unpaired Mann–Whitney test. (B) Immunoblot for inflammasome component expression in whole-cell extracts (4 h LPS). (C) Cell lysates and cell-free supernatants were harvested at various times after nigericin stimulation (0, 2, 4, 8 h), and analyzed by Western blot. *, Nonspecific band. (D) bVAD-fmk was applied at the indicated time points. Supernatant was removed, and cells were lysed 8 h after nigericin stimulation, and active caspase-1 was pulled down using streptavidin beads. Streptavidin-bound and -unbound fractions from mixed supernatants/extracts were analyzed by Western blot. All data are representative of at least three (A, B, C [WT], D [WT]) or two (C [ Ice −/− ], D [ Ice −/− ]) independent experiments.

    Techniques Used: Activity Assay, Microscopy, MANN-WHITNEY, Expressing, Western Blot

    Caspase-1 activity localizes to the inflammasome. (A) Mouse macrophages were either left untreated or LPS-primed for 4 h before nigericin stimulation for a further 2 h. bVAD-fmk was applied to cells at various times after nigericin (0, 15, 30, 60 min). Cell supernatants were removed and subjected to streptavidin pull-down, which failed to detect active caspase-1 species in this fraction (see Fig. S3 A for pull-down analysis of cell culture medium). Cells were harvested and fractionated. The ASC speck-containing (Triton X-100–insoluble) fraction and the Triton X-100–soluble fraction were analyzed for caspase-1 species by Western blot. Cells were treated with 5 mM glycine 30 min before nigericin to delay cell rupture (right). (B) Macrophages were LPS-primed (4 h) and stimulated with nigericin for 30 min (with and without bVAD-fmk application immediately before nigericin). ASC was immunoprecipitated, and coimmunoprecipitated caspase-1 species were examined by immunoblot. *, Nonspecific band. (C) Macrophages were LPS-primed (4 h) and stimulated with nigericin for 30 min. The FAM-FLICA caspase-1 activity probe was added at the indicated times after nigericin stimulation to localize caspase-1 activity relative to ASC. (D and E) LPS-primed WT or Casp1- deficient Ice −/− macrophages were stimulated with nigericin. Caspase inhibitors (bVAD-fmk caspase activity probe, VX765) were applied at the indicated times after nigericin stimulation, with (D) or without (E) 5 mM glycine in the cell culture media. 30 min after nigericin stimulation, cells were fixed and PLA was performed to detect active caspase-1 (α-biotin/α-caspase-1 LS; (D) or caspase-1 dimers (α-caspase-1 LS:PLUS/α-caspase-1 LS:MINUS; E). DAPI labeled the nucleus. All data are representative of at least three (A–D) or two (E) independent experiments. Arrowheads indicate foci of caspase-1 activity or dimers.
    Figure Legend Snippet: Caspase-1 activity localizes to the inflammasome. (A) Mouse macrophages were either left untreated or LPS-primed for 4 h before nigericin stimulation for a further 2 h. bVAD-fmk was applied to cells at various times after nigericin (0, 15, 30, 60 min). Cell supernatants were removed and subjected to streptavidin pull-down, which failed to detect active caspase-1 species in this fraction (see Fig. S3 A for pull-down analysis of cell culture medium). Cells were harvested and fractionated. The ASC speck-containing (Triton X-100–insoluble) fraction and the Triton X-100–soluble fraction were analyzed for caspase-1 species by Western blot. Cells were treated with 5 mM glycine 30 min before nigericin to delay cell rupture (right). (B) Macrophages were LPS-primed (4 h) and stimulated with nigericin for 30 min (with and without bVAD-fmk application immediately before nigericin). ASC was immunoprecipitated, and coimmunoprecipitated caspase-1 species were examined by immunoblot. *, Nonspecific band. (C) Macrophages were LPS-primed (4 h) and stimulated with nigericin for 30 min. The FAM-FLICA caspase-1 activity probe was added at the indicated times after nigericin stimulation to localize caspase-1 activity relative to ASC. (D and E) LPS-primed WT or Casp1- deficient Ice −/− macrophages were stimulated with nigericin. Caspase inhibitors (bVAD-fmk caspase activity probe, VX765) were applied at the indicated times after nigericin stimulation, with (D) or without (E) 5 mM glycine in the cell culture media. 30 min after nigericin stimulation, cells were fixed and PLA was performed to detect active caspase-1 (α-biotin/α-caspase-1 LS; (D) or caspase-1 dimers (α-caspase-1 LS:PLUS/α-caspase-1 LS:MINUS; E). DAPI labeled the nucleus. All data are representative of at least three (A–D) or two (E) independent experiments. Arrowheads indicate foci of caspase-1 activity or dimers.

    Techniques Used: Activity Assay, Cell Culture, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Labeling

    Inflammasome size specifies active caspase-1 species and activity duration. (A–C) LPS-primed macrophages were transfected with flagellin (A) or stimulated with nigericin (B and C). bVAD-fmk was applied at the indicated time points (A and B). (A and B) Pull-down of active caspase-1 from macrophages. 1% IGEPAL was added to macrophages at 3 h after flagellin and 2 h after nigericin stimulation, to lyse cells directly in their culture medium. Streptavidin-coated beads pulled down active caspase-1 bound to the biotin-labeled activity probe in mixed lysates/supernatants. (C) ASC speck intensity of Asc +/+ and Asc +/− macrophages (4 h LPS + nigericin, with VX765 added 1 h before nigericin to prevent cell death), quantified from microscopy images. Each circle is one speck ( n = 40–55), and significance between samples was assessed by nonparametric, unpaired Mann–Whitney test. All data are representative of at least three (A and B) or two (C) independent experiments.
    Figure Legend Snippet: Inflammasome size specifies active caspase-1 species and activity duration. (A–C) LPS-primed macrophages were transfected with flagellin (A) or stimulated with nigericin (B and C). bVAD-fmk was applied at the indicated time points (A and B). (A and B) Pull-down of active caspase-1 from macrophages. 1% IGEPAL was added to macrophages at 3 h after flagellin and 2 h after nigericin stimulation, to lyse cells directly in their culture medium. Streptavidin-coated beads pulled down active caspase-1 bound to the biotin-labeled activity probe in mixed lysates/supernatants. (C) ASC speck intensity of Asc +/+ and Asc +/− macrophages (4 h LPS + nigericin, with VX765 added 1 h before nigericin to prevent cell death), quantified from microscopy images. Each circle is one speck ( n = 40–55), and significance between samples was assessed by nonparametric, unpaired Mann–Whitney test. All data are representative of at least three (A and B) or two (C) independent experiments.

    Techniques Used: Activity Assay, Transfection, Labeling, Microscopy, MANN-WHITNEY

    Active caspase-1 is predominantly a transient p33/p10 species in nigericin-stimulated macrophages. (A) Representation of potential self-processing sites within the CDL and IDL of mouse caspase-1, relative to the catalytic cysteine (C284). (B) Possible species of dimeric caspase-1 generated by CDL and/or IDL cleavage. (C) Pull-down of active caspase-1 from mouse macrophages, using the bVAD-fmk caspase activity probe. Macrophages were left untreated or primed with LPS for 4 h, and then stimulated with nigericin for a further 4 h before addition of 1% IGEPAL into the well, to lyse cells directly in their culture medium. bVAD-fmk was applied to cells 1 h before (−1 h), during (0 h), or after (0.5, 1, 3, 4 h) nigericin addition. Streptavidin-coated beads pulled down active caspase-1 bound to the biotin-labeled activity probe in mixed lysates/supernatants. Streptavidin-bound and -unbound fractions were analyzed by Western blot using antibodies directed against the caspase-1 large and small subunits (LS, SS). (D) LPS-primed WT and Gsdmd −/− macrophages were stimulated with nigericin and harvested at various time points until 2 h after nigericin stimulation. Cell supernatants were precipitated and resuspended in cell extracts, and the kinetics of caspase-1 substrate cleavage (pro-IL-1β cleavage to p17, pro-GSDMD to GSDMD p30) was examined by Western blot. All data are representative of at least three independent experiments.
    Figure Legend Snippet: Active caspase-1 is predominantly a transient p33/p10 species in nigericin-stimulated macrophages. (A) Representation of potential self-processing sites within the CDL and IDL of mouse caspase-1, relative to the catalytic cysteine (C284). (B) Possible species of dimeric caspase-1 generated by CDL and/or IDL cleavage. (C) Pull-down of active caspase-1 from mouse macrophages, using the bVAD-fmk caspase activity probe. Macrophages were left untreated or primed with LPS for 4 h, and then stimulated with nigericin for a further 4 h before addition of 1% IGEPAL into the well, to lyse cells directly in their culture medium. bVAD-fmk was applied to cells 1 h before (−1 h), during (0 h), or after (0.5, 1, 3, 4 h) nigericin addition. Streptavidin-coated beads pulled down active caspase-1 bound to the biotin-labeled activity probe in mixed lysates/supernatants. Streptavidin-bound and -unbound fractions were analyzed by Western blot using antibodies directed against the caspase-1 large and small subunits (LS, SS). (D) LPS-primed WT and Gsdmd −/− macrophages were stimulated with nigericin and harvested at various time points until 2 h after nigericin stimulation. Cell supernatants were precipitated and resuspended in cell extracts, and the kinetics of caspase-1 substrate cleavage (pro-IL-1β cleavage to p17, pro-GSDMD to GSDMD p30) was examined by Western blot. All data are representative of at least three independent experiments.

    Techniques Used: Generated, Activity Assay, Labeling, Western Blot

    Summary model for mechanisms by which inflammasomes direct the active caspase-1 species and duration of caspase-1 activity. See Discussion for details.
    Figure Legend Snippet: Summary model for mechanisms by which inflammasomes direct the active caspase-1 species and duration of caspase-1 activity. See Discussion for details.

    Techniques Used: Activity Assay

    12) Product Images from "Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity"

    Article Title: Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20172222

    Caspase-1 processing at the CDL destabilizes caspase-1 dimers and terminates protease activity. Bone marrow progenitors from Ice −/− ( Casp1 −/− /Casp11 null/null ) mice were retrovirally reconstituted for mouse caspase-1 (WT, C284 catalytic mutant, or CDL-uncleavable mutant) during their differentiation to macrophages. (A) Differentiated macrophages were then left untreated or LPS-treated for 6 h, or were LPS-primed for 4 h before stimulation with nigericin for 0.5, 1, or 2 h. Cell extracts and cell-free supernatants were mixed and analyzed by immunoblot. (B) Differentiated, transduced macrophages were LPS-primed for 4 h, before stimulation with nigericin for 30 min. Cell culture medium was then replaced with caspase assay buffer containing the caspase-1 fluorogenic substrate, YVAD-afc, and caspase-1–like activity was monitored over the next 30 min. (C) An engineered caspase-1 system allowed controlled caspase-1 dimerization by AP20187 (AP) and CDL cleavage by thrombin (thr). 1 µM AP20187 was added to HEK293T-expressing caspase-1 constructs (WT vs. catalytic mutant, C284A) for 10 min. Cell culture medium was replaced for caspase activity buffer supplemented with digitonin to lyse cells for activity assays (± 20 U/ml thrombin). (D) Immunoblot analysis of caspase-1 cleavage by thrombin after incubation for 1 h. (E) Caspase activity assay, in which thrombin was added to cell lysates at the same time as the YVAD-afc fluorogenic substrate. (F) Cleavage assay in which thrombin was added to caspase-1–expressing cell lysates at the same time as natural substrates (lysates from HEK293T ectopically expressing V5-tagged substrates) and incubated for 0.5 h. (G) Thrombin was added to caspase-1_C284A–expressing cell lysates and incubated for 1 h. Bismaleimidohexane was then added to lysates for 1 h to cross-link caspase-1 dimers. Graphs are mean + SD of triplicate wells from a single experiment. All data are representative of at least two (G) or three (A–F) independent experiments.
    Figure Legend Snippet: Caspase-1 processing at the CDL destabilizes caspase-1 dimers and terminates protease activity. Bone marrow progenitors from Ice −/− ( Casp1 −/− /Casp11 null/null ) mice were retrovirally reconstituted for mouse caspase-1 (WT, C284 catalytic mutant, or CDL-uncleavable mutant) during their differentiation to macrophages. (A) Differentiated macrophages were then left untreated or LPS-treated for 6 h, or were LPS-primed for 4 h before stimulation with nigericin for 0.5, 1, or 2 h. Cell extracts and cell-free supernatants were mixed and analyzed by immunoblot. (B) Differentiated, transduced macrophages were LPS-primed for 4 h, before stimulation with nigericin for 30 min. Cell culture medium was then replaced with caspase assay buffer containing the caspase-1 fluorogenic substrate, YVAD-afc, and caspase-1–like activity was monitored over the next 30 min. (C) An engineered caspase-1 system allowed controlled caspase-1 dimerization by AP20187 (AP) and CDL cleavage by thrombin (thr). 1 µM AP20187 was added to HEK293T-expressing caspase-1 constructs (WT vs. catalytic mutant, C284A) for 10 min. Cell culture medium was replaced for caspase activity buffer supplemented with digitonin to lyse cells for activity assays (± 20 U/ml thrombin). (D) Immunoblot analysis of caspase-1 cleavage by thrombin after incubation for 1 h. (E) Caspase activity assay, in which thrombin was added to cell lysates at the same time as the YVAD-afc fluorogenic substrate. (F) Cleavage assay in which thrombin was added to caspase-1–expressing cell lysates at the same time as natural substrates (lysates from HEK293T ectopically expressing V5-tagged substrates) and incubated for 0.5 h. (G) Thrombin was added to caspase-1_C284A–expressing cell lysates and incubated for 1 h. Bismaleimidohexane was then added to lysates for 1 h to cross-link caspase-1 dimers. Graphs are mean + SD of triplicate wells from a single experiment. All data are representative of at least two (G) or three (A–F) independent experiments.

    Techniques Used: Activity Assay, Mouse Assay, Mutagenesis, Cell Culture, Caspase Assay, Expressing, Construct, Incubation, Caspase Activity Assay, Cleavage Assay

    Caspase-1 activity localizes to the inflammasome. (A) Mouse macrophages were either left untreated or LPS-primed for 4 h before nigericin stimulation for a further 2 h. bVAD-fmk was applied to cells at various times after nigericin (0, 15, 30, 60 min). Cell supernatants were removed and subjected to streptavidin pull-down, which failed to detect active caspase-1 species in this fraction (see Fig. S3 A for pull-down analysis of cell culture medium). Cells were harvested and fractionated. The ASC speck-containing (Triton X-100–insoluble) fraction and the Triton X-100–soluble fraction were analyzed for caspase-1 species by Western blot. Cells were treated with 5 mM glycine 30 min before nigericin to delay cell rupture (right). (B) Macrophages were LPS-primed (4 h) and stimulated with nigericin for 30 min (with and without bVAD-fmk application immediately before nigericin). ASC was immunoprecipitated, and coimmunoprecipitated caspase-1 species were examined by immunoblot. *, Nonspecific band. (C) Macrophages were LPS-primed (4 h) and stimulated with nigericin for 30 min. The FAM-FLICA caspase-1 activity probe was added at the indicated times after nigericin stimulation to localize caspase-1 activity relative to ASC. (D and E) LPS-primed WT or Casp1- deficient Ice −/− macrophages were stimulated with nigericin. Caspase inhibitors (bVAD-fmk caspase activity probe, VX765) were applied at the indicated times after nigericin stimulation, with (D) or without (E) 5 mM glycine in the cell culture media. 30 min after nigericin stimulation, cells were fixed and PLA was performed to detect active caspase-1 (α-biotin/α-caspase-1 LS; (D) or caspase-1 dimers (α-caspase-1 LS:PLUS/α-caspase-1 LS:MINUS; E). DAPI labeled the nucleus. All data are representative of at least three (A–D) or two (E) independent experiments. Arrowheads indicate foci of caspase-1 activity or dimers.
    Figure Legend Snippet: Caspase-1 activity localizes to the inflammasome. (A) Mouse macrophages were either left untreated or LPS-primed for 4 h before nigericin stimulation for a further 2 h. bVAD-fmk was applied to cells at various times after nigericin (0, 15, 30, 60 min). Cell supernatants were removed and subjected to streptavidin pull-down, which failed to detect active caspase-1 species in this fraction (see Fig. S3 A for pull-down analysis of cell culture medium). Cells were harvested and fractionated. The ASC speck-containing (Triton X-100–insoluble) fraction and the Triton X-100–soluble fraction were analyzed for caspase-1 species by Western blot. Cells were treated with 5 mM glycine 30 min before nigericin to delay cell rupture (right). (B) Macrophages were LPS-primed (4 h) and stimulated with nigericin for 30 min (with and without bVAD-fmk application immediately before nigericin). ASC was immunoprecipitated, and coimmunoprecipitated caspase-1 species were examined by immunoblot. *, Nonspecific band. (C) Macrophages were LPS-primed (4 h) and stimulated with nigericin for 30 min. The FAM-FLICA caspase-1 activity probe was added at the indicated times after nigericin stimulation to localize caspase-1 activity relative to ASC. (D and E) LPS-primed WT or Casp1- deficient Ice −/− macrophages were stimulated with nigericin. Caspase inhibitors (bVAD-fmk caspase activity probe, VX765) were applied at the indicated times after nigericin stimulation, with (D) or without (E) 5 mM glycine in the cell culture media. 30 min after nigericin stimulation, cells were fixed and PLA was performed to detect active caspase-1 (α-biotin/α-caspase-1 LS; (D) or caspase-1 dimers (α-caspase-1 LS:PLUS/α-caspase-1 LS:MINUS; E). DAPI labeled the nucleus. All data are representative of at least three (A–D) or two (E) independent experiments. Arrowheads indicate foci of caspase-1 activity or dimers.

    Techniques Used: Activity Assay, Cell Culture, Western Blot, Immunoprecipitation, Proximity Ligation Assay, Labeling

    Cell type specifies caspase-1 activity duration. (A–D) LPS-primed (A and B) macrophages or (A–D) neutrophils were left untreated or stimulated with nigericin. (A) ASC speck intensity of Ice −/− macrophages and neutrophils (4 h LPS + nigericin), quantified from microscopy images. Each circle is one speck ( n = 20–55), and significance between samples was assessed by nonparametric, unpaired Mann–Whitney test. (B) Immunoblot for inflammasome component expression in whole-cell extracts (4 h LPS). (C) Cell lysates and cell-free supernatants were harvested at various times after nigericin stimulation (0, 2, 4, 8 h), and analyzed by Western blot. *, Nonspecific band. (D) bVAD-fmk was applied at the indicated time points. Supernatant was removed, and cells were lysed 8 h after nigericin stimulation, and active caspase-1 was pulled down using streptavidin beads. Streptavidin-bound and -unbound fractions from mixed supernatants/extracts were analyzed by Western blot. All data are representative of at least three (A, B, C [WT], D [WT]) or two (C [ Ice −/− ], D [ Ice −/− ]) independent experiments.
    Figure Legend Snippet: Cell type specifies caspase-1 activity duration. (A–D) LPS-primed (A and B) macrophages or (A–D) neutrophils were left untreated or stimulated with nigericin. (A) ASC speck intensity of Ice −/− macrophages and neutrophils (4 h LPS + nigericin), quantified from microscopy images. Each circle is one speck ( n = 20–55), and significance between samples was assessed by nonparametric, unpaired Mann–Whitney test. (B) Immunoblot for inflammasome component expression in whole-cell extracts (4 h LPS). (C) Cell lysates and cell-free supernatants were harvested at various times after nigericin stimulation (0, 2, 4, 8 h), and analyzed by Western blot. *, Nonspecific band. (D) bVAD-fmk was applied at the indicated time points. Supernatant was removed, and cells were lysed 8 h after nigericin stimulation, and active caspase-1 was pulled down using streptavidin beads. Streptavidin-bound and -unbound fractions from mixed supernatants/extracts were analyzed by Western blot. All data are representative of at least three (A, B, C [WT], D [WT]) or two (C [ Ice −/− ], D [ Ice −/− ]) independent experiments.

    Techniques Used: Activity Assay, Microscopy, MANN-WHITNEY, Expressing, Western Blot

    Inflammasome size specifies active caspase-1 species and activity duration. (A–C) LPS-primed macrophages were transfected with flagellin (A) or stimulated with nigericin (B and C). bVAD-fmk was applied at the indicated time points (A and B). (A and B) Pull-down of active caspase-1 from macrophages. 1% IGEPAL was added to macrophages at 3 h after flagellin and 2 h after nigericin stimulation, to lyse cells directly in their culture medium. Streptavidin-coated beads pulled down active caspase-1 bound to the biotin-labeled activity probe in mixed lysates/supernatants. (C) ASC speck intensity of Asc +/+ and Asc +/− macrophages (4 h LPS + nigericin, with VX765 added 1 h before nigericin to prevent cell death), quantified from microscopy images. Each circle is one speck ( n = 40–55), and significance between samples was assessed by nonparametric, unpaired Mann–Whitney test. All data are representative of at least three (A and B) or two (C) independent experiments.
    Figure Legend Snippet: Inflammasome size specifies active caspase-1 species and activity duration. (A–C) LPS-primed macrophages were transfected with flagellin (A) or stimulated with nigericin (B and C). bVAD-fmk was applied at the indicated time points (A and B). (A and B) Pull-down of active caspase-1 from macrophages. 1% IGEPAL was added to macrophages at 3 h after flagellin and 2 h after nigericin stimulation, to lyse cells directly in their culture medium. Streptavidin-coated beads pulled down active caspase-1 bound to the biotin-labeled activity probe in mixed lysates/supernatants. (C) ASC speck intensity of Asc +/+ and Asc +/− macrophages (4 h LPS + nigericin, with VX765 added 1 h before nigericin to prevent cell death), quantified from microscopy images. Each circle is one speck ( n = 40–55), and significance between samples was assessed by nonparametric, unpaired Mann–Whitney test. All data are representative of at least three (A and B) or two (C) independent experiments.

    Techniques Used: Activity Assay, Transfection, Labeling, Microscopy, MANN-WHITNEY

    Active caspase-1 is predominantly a transient p33/p10 species in nigericin-stimulated macrophages. (A) Representation of potential self-processing sites within the CDL and IDL of mouse caspase-1, relative to the catalytic cysteine (C284). (B) Possible species of dimeric caspase-1 generated by CDL and/or IDL cleavage. (C) Pull-down of active caspase-1 from mouse macrophages, using the bVAD-fmk caspase activity probe. Macrophages were left untreated or primed with LPS for 4 h, and then stimulated with nigericin for a further 4 h before addition of 1% IGEPAL into the well, to lyse cells directly in their culture medium. bVAD-fmk was applied to cells 1 h before (−1 h), during (0 h), or after (0.5, 1, 3, 4 h) nigericin addition. Streptavidin-coated beads pulled down active caspase-1 bound to the biotin-labeled activity probe in mixed lysates/supernatants. Streptavidin-bound and -unbound fractions were analyzed by Western blot using antibodies directed against the caspase-1 large and small subunits (LS, SS). (D) LPS-primed WT and Gsdmd −/− macrophages were stimulated with nigericin and harvested at various time points until 2 h after nigericin stimulation. Cell supernatants were precipitated and resuspended in cell extracts, and the kinetics of caspase-1 substrate cleavage (pro-IL-1β cleavage to p17, pro-GSDMD to GSDMD p30) was examined by Western blot. All data are representative of at least three independent experiments.
    Figure Legend Snippet: Active caspase-1 is predominantly a transient p33/p10 species in nigericin-stimulated macrophages. (A) Representation of potential self-processing sites within the CDL and IDL of mouse caspase-1, relative to the catalytic cysteine (C284). (B) Possible species of dimeric caspase-1 generated by CDL and/or IDL cleavage. (C) Pull-down of active caspase-1 from mouse macrophages, using the bVAD-fmk caspase activity probe. Macrophages were left untreated or primed with LPS for 4 h, and then stimulated with nigericin for a further 4 h before addition of 1% IGEPAL into the well, to lyse cells directly in their culture medium. bVAD-fmk was applied to cells 1 h before (−1 h), during (0 h), or after (0.5, 1, 3, 4 h) nigericin addition. Streptavidin-coated beads pulled down active caspase-1 bound to the biotin-labeled activity probe in mixed lysates/supernatants. Streptavidin-bound and -unbound fractions were analyzed by Western blot using antibodies directed against the caspase-1 large and small subunits (LS, SS). (D) LPS-primed WT and Gsdmd −/− macrophages were stimulated with nigericin and harvested at various time points until 2 h after nigericin stimulation. Cell supernatants were precipitated and resuspended in cell extracts, and the kinetics of caspase-1 substrate cleavage (pro-IL-1β cleavage to p17, pro-GSDMD to GSDMD p30) was examined by Western blot. All data are representative of at least three independent experiments.

    Techniques Used: Generated, Activity Assay, Labeling, Western Blot

    Summary model for mechanisms by which inflammasomes direct the active caspase-1 species and duration of caspase-1 activity. See Discussion for details.
    Figure Legend Snippet: Summary model for mechanisms by which inflammasomes direct the active caspase-1 species and duration of caspase-1 activity. See Discussion for details.

    Techniques Used: Activity Assay

    13) Product Images from "Small Molecular TRAIL Inducer ONC201 Induces Death in Lung Cancer Cells: A Preclinical Study"

    Article Title: Small Molecular TRAIL Inducer ONC201 Induces Death in Lung Cancer Cells: A Preclinical Study

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0162133

    ONC201 provokes apoptosis in human lung cancer cells. A549 (A-F), H460 (G-I) or primary human lung cancer cells (“Pat-1/-2”) (G-I), as well as the lung epithelial BEAS-2B cells (G-I) were treated with applied concentration of ONC201 for indicated time, TRAIL and DR5 expressions were tested by real-time PCR assay (A, B, G and H) or Western blot assay (B, Upper panel); Relative caspase-8/-3 activity was also presented (C and D); Cell apoptosis was tested by ssDNA ELISA assay (E) and Sub-G1 FACS assay (F and I). The results presented were representative of three independent experiments. The values were expressed as the means ± SD. “C” stands for untreated control group. * p
    Figure Legend Snippet: ONC201 provokes apoptosis in human lung cancer cells. A549 (A-F), H460 (G-I) or primary human lung cancer cells (“Pat-1/-2”) (G-I), as well as the lung epithelial BEAS-2B cells (G-I) were treated with applied concentration of ONC201 for indicated time, TRAIL and DR5 expressions were tested by real-time PCR assay (A, B, G and H) or Western blot assay (B, Upper panel); Relative caspase-8/-3 activity was also presented (C and D); Cell apoptosis was tested by ssDNA ELISA assay (E) and Sub-G1 FACS assay (F and I). The results presented were representative of three independent experiments. The values were expressed as the means ± SD. “C” stands for untreated control group. * p

    Techniques Used: Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, FACS

    14) Product Images from "Ceramide Production Mediates Aldosterone-Induced Human Umbilical Vein Endothelial Cell (HUVEC) Damages"

    Article Title: Ceramide Production Mediates Aldosterone-Induced Human Umbilical Vein Endothelial Cell (HUVEC) Damages

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0146944

    Aldosterone induces caspase-3-dependent apoptotic death in HUVECs. HUVECs were treated with applied concentrations of aldosterone (1–1000 nM) for indicate time, cell apoptosis was evidenced by Annexin V FACS assay (A, representative FACS images were shown in lower panel), histone DNA ELISA assay (B), caspase-3 activity assay (C, lower panel) and Western blot assaying of cleavead-caspase-3 (“C-Caspase-3”) (C, upper panel). HUVECs, pre-treated with the caspase-3 inhibitor z-DVED-fmk (“ZDVED”, 25 μM)/AC-DEVD-CHO (“AC-DEVD”, 25 μM), or the pan caspase inhibitor z-VAD-fmk (“ZVAD”, 25 μM) for 1 hour, were stimulated with aldosterone (100 nM), caspase-3 activity and cell apoptosis were analyzed by the caspase-3 activity assay (D) and Annexin V FACS assay (E), respectively; Cell survival was tested by the MTT assay (F). Data were expressed as the mean ± SD. For each assay, n = 5. Experiments in this figure were repeated three times, and similar results were obtained. * p
    Figure Legend Snippet: Aldosterone induces caspase-3-dependent apoptotic death in HUVECs. HUVECs were treated with applied concentrations of aldosterone (1–1000 nM) for indicate time, cell apoptosis was evidenced by Annexin V FACS assay (A, representative FACS images were shown in lower panel), histone DNA ELISA assay (B), caspase-3 activity assay (C, lower panel) and Western blot assaying of cleavead-caspase-3 (“C-Caspase-3”) (C, upper panel). HUVECs, pre-treated with the caspase-3 inhibitor z-DVED-fmk (“ZDVED”, 25 μM)/AC-DEVD-CHO (“AC-DEVD”, 25 μM), or the pan caspase inhibitor z-VAD-fmk (“ZVAD”, 25 μM) for 1 hour, were stimulated with aldosterone (100 nM), caspase-3 activity and cell apoptosis were analyzed by the caspase-3 activity assay (D) and Annexin V FACS assay (E), respectively; Cell survival was tested by the MTT assay (F). Data were expressed as the mean ± SD. For each assay, n = 5. Experiments in this figure were repeated three times, and similar results were obtained. * p

    Techniques Used: FACS, Enzyme-linked Immunosorbent Assay, Caspase-3 Activity Assay, Western Blot, Activity Assay, MTT Assay

    Aldosterone mainly induces C18 ceramide production in HUVECs. HUVECs were treated with applied concentrations of aldosterone (1–1000 nM) for 4 hours, total cellular ceramide level was analyzed by the DAG kinase assay, and was normalized to the untreated control (“Ctrl”) group (A), individual ceramide level was detected by LS-MS assay as described (F). HUVECs, pretreated with PDMP (10 μM), S1P (10 μM) or C6 ceramide (25 μM) for 1 hour, were stimulated with aldosterone (100 nM), cellular ceramide was analyzed (B); Cell survival was tested by MTT assay (C), and cell apoptosis was tested by the caspase-3 activity assay (D) or the Histone DNA ELISA assay (E). For each assay, n = 3. Experiments in this figure were repeated three times, and similar results were obtained. * p
    Figure Legend Snippet: Aldosterone mainly induces C18 ceramide production in HUVECs. HUVECs were treated with applied concentrations of aldosterone (1–1000 nM) for 4 hours, total cellular ceramide level was analyzed by the DAG kinase assay, and was normalized to the untreated control (“Ctrl”) group (A), individual ceramide level was detected by LS-MS assay as described (F). HUVECs, pretreated with PDMP (10 μM), S1P (10 μM) or C6 ceramide (25 μM) for 1 hour, were stimulated with aldosterone (100 nM), cellular ceramide was analyzed (B); Cell survival was tested by MTT assay (C), and cell apoptosis was tested by the caspase-3 activity assay (D) or the Histone DNA ELISA assay (E). For each assay, n = 3. Experiments in this figure were repeated three times, and similar results were obtained. * p

    Techniques Used: Kinase Assay, Mass Spectrometry, MTT Assay, Caspase-3 Activity Assay, Enzyme-linked Immunosorbent Assay

    Ceramide synthase 1 mediates aldosterone-induced C18 ceramide production and following HUVEC damages. Expression of CerS-1 and tubulin (the equal loading) in HUVECs expressing scramble control shRNA or CerS-1 shRNA (two colonies) was shown (A), CerS-1 expression (vs. tubulin) was quantified (A). Above cells were treated with or without aldosterone (100 nM), C18 and C24 ceramide production (B and C), cell survival (D) and cell apoptosis (Caspase-3 activity, E and Histone DNA EILSA OD, F) were tested. Expression of CerS-1 and β-actin (the equal loading) in HUVECs expressing CerS-1-cDNA or the empty vector (p-Super-puro) was shown, CerS-1 expression (vs. β-actin) was quantified (G); Aldo (100 nM)-induced C18 ceramide production (H, 4 hours), cell viability reduction (I, 24 hours), and caspase-3 activity (J, 12 hours) in above cells were tested. For each assay, n = 3. Experiments in this figure were repeated three times, and similar results were obtained. * p
    Figure Legend Snippet: Ceramide synthase 1 mediates aldosterone-induced C18 ceramide production and following HUVEC damages. Expression of CerS-1 and tubulin (the equal loading) in HUVECs expressing scramble control shRNA or CerS-1 shRNA (two colonies) was shown (A), CerS-1 expression (vs. tubulin) was quantified (A). Above cells were treated with or without aldosterone (100 nM), C18 and C24 ceramide production (B and C), cell survival (D) and cell apoptosis (Caspase-3 activity, E and Histone DNA EILSA OD, F) were tested. Expression of CerS-1 and β-actin (the equal loading) in HUVECs expressing CerS-1-cDNA or the empty vector (p-Super-puro) was shown, CerS-1 expression (vs. β-actin) was quantified (G); Aldo (100 nM)-induced C18 ceramide production (H, 4 hours), cell viability reduction (I, 24 hours), and caspase-3 activity (J, 12 hours) in above cells were tested. For each assay, n = 3. Experiments in this figure were repeated three times, and similar results were obtained. * p

    Techniques Used: Expressing, shRNA, Activity Assay, Plasmid Preparation

    Eplerenone blocks aldosterone-induced ceramide production and following HUVEC cytotoxicity. HUVECs, pretreated with Eplerenone (10 μM) for 1 hour, were stimulated with aldosterone (100 nM), C18 ceramide (A, LS-MS assay), C24 ceramide (B, LS-MS assay), and total ceramide (C, DAG kinase assay) were analyzed after 4 hours; Cell viability was tested by MTT assay (D), and cell apoptosis was tested by the caspase-3 activity assay (E) and Histone DNA ELISA assay (F). For each assay, n = 3. Experiments in this figure were repeated three times, and similar results were obtained. * p
    Figure Legend Snippet: Eplerenone blocks aldosterone-induced ceramide production and following HUVEC cytotoxicity. HUVECs, pretreated with Eplerenone (10 μM) for 1 hour, were stimulated with aldosterone (100 nM), C18 ceramide (A, LS-MS assay), C24 ceramide (B, LS-MS assay), and total ceramide (C, DAG kinase assay) were analyzed after 4 hours; Cell viability was tested by MTT assay (D), and cell apoptosis was tested by the caspase-3 activity assay (E) and Histone DNA ELISA assay (F). For each assay, n = 3. Experiments in this figure were repeated three times, and similar results were obtained. * p

    Techniques Used: Mass Spectrometry, Kinase Assay, MTT Assay, Caspase-3 Activity Assay, Enzyme-linked Immunosorbent Assay

    15) Product Images from "Wip1 suppresses apoptotic cell death through direct dephosphorylation of BAX in response to γ-radiation"

    Article Title: Wip1 suppresses apoptotic cell death through direct dephosphorylation of BAX in response to γ-radiation

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.252

    Depletion of Wip1 enhances IR-induced apoptotic cell death. ( a ) HeLa or LNCaP cells were irradiated with 10 Gy. After 72 h, the cells were harvested and stained with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC). The proportions of apoptotic cells within the annexin V-FITC and PI-positive subpopulations were determined using fluorescence-activated cell sorting (FACS). ( b ) LNCaP cells were transfected with 50 nM of either control small interfering (si)RNA or Wip1 siRNA. At 48 h post-transfection, the cells were irradiated with 10 Gy. After 72 h, the cells were stained with PI and annexin V-FITC. Apoptotic cell death was determined by FACS. ( c ) LNCaP cells were transfected with 50 nM of either control siRNA or Wip1 siRNA for 36 h, and then irradiated with 10 Gy. After 40 h, the transfectants were assayed for caspase-3 activity. All assays were performed in triplicate. The data show the mean±S.D. (* P
    Figure Legend Snippet: Depletion of Wip1 enhances IR-induced apoptotic cell death. ( a ) HeLa or LNCaP cells were irradiated with 10 Gy. After 72 h, the cells were harvested and stained with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC). The proportions of apoptotic cells within the annexin V-FITC and PI-positive subpopulations were determined using fluorescence-activated cell sorting (FACS). ( b ) LNCaP cells were transfected with 50 nM of either control small interfering (si)RNA or Wip1 siRNA. At 48 h post-transfection, the cells were irradiated with 10 Gy. After 72 h, the cells were stained with PI and annexin V-FITC. Apoptotic cell death was determined by FACS. ( c ) LNCaP cells were transfected with 50 nM of either control siRNA or Wip1 siRNA for 36 h, and then irradiated with 10 Gy. After 40 h, the transfectants were assayed for caspase-3 activity. All assays were performed in triplicate. The data show the mean±S.D. (* P

    Techniques Used: Irradiation, Staining, Fluorescence, FACS, Transfection, Activity Assay

    Wip1 dephosphorylates active BAX and suppresses mitochondria-dependent apoptosis. ( a ) Recombinant Wip1 does not dephosphorylate BAX-derived phosphopeptides encompassing Ser87, Ser163, Ser 184, and Thr167, as shown in an in vitro phosphatase assay. Released free phosphate was measured by absorbance at 630 nm in the presence of molybdate dye. Phosphopeptides p180T from p38 MAPK and p31T from UNG2 were used as positive and negative controls, respectively. 10 ( b ) Phosphorylation sites on Ser and Thr residues in BAX were predicted with the NetPhos 2.0 server. 30 ( c ) Expression vectors carrying GFP wild-type or one of nine mutated GFP-BAX mutant constructs were transfected into HeLa cells. At 48 h post-transfection, the cells were exposed to γ -radiation and then analyzed 24 h later. BAX translocation to the mitochondria results in intense green fluorescence (phosphothreonine and phosphoserine are indicated in bold). ( d ) BAX p172T, p174T, and p186T phosphopeptides were incubated with recombinant Wip1 protein. Free phosphate released by dephosphorylation was measured. Two polypeptides of p38 MAPK p180T served as the positive control, and UNG2 p31T as the negative control. 10 Assays were also performed in the absence of magnesium or Wip1 and in the presence of okadaic acid. Wip1 phosphatase activity is magnesium-dependent and insensitive to okadaic acid. ( e ) DU145 cells were transfected with BAX or mutated BAX (T172D, T174D, and T186D) expression plasmids. After 48 h, the transfectants were irradiated, and then harvested, and finally assayed for caspase-3 activity. ( f ) DU145 cells were transfected with wild-type or mutated BAX together with wild-type Wip1 or phosphatase-dead (PD) Wip1 (D314A and D105A) expression vectors for 48 h and then irradiated with 10 Gy. After 24 h, the transfectants were assayed for caspase-3 activity. The mean of three experiments is shown in each column; bars in ( d ), ( e ), and ( f ) correspond to the S.D. Asterisks (*) indicate P
    Figure Legend Snippet: Wip1 dephosphorylates active BAX and suppresses mitochondria-dependent apoptosis. ( a ) Recombinant Wip1 does not dephosphorylate BAX-derived phosphopeptides encompassing Ser87, Ser163, Ser 184, and Thr167, as shown in an in vitro phosphatase assay. Released free phosphate was measured by absorbance at 630 nm in the presence of molybdate dye. Phosphopeptides p180T from p38 MAPK and p31T from UNG2 were used as positive and negative controls, respectively. 10 ( b ) Phosphorylation sites on Ser and Thr residues in BAX were predicted with the NetPhos 2.0 server. 30 ( c ) Expression vectors carrying GFP wild-type or one of nine mutated GFP-BAX mutant constructs were transfected into HeLa cells. At 48 h post-transfection, the cells were exposed to γ -radiation and then analyzed 24 h later. BAX translocation to the mitochondria results in intense green fluorescence (phosphothreonine and phosphoserine are indicated in bold). ( d ) BAX p172T, p174T, and p186T phosphopeptides were incubated with recombinant Wip1 protein. Free phosphate released by dephosphorylation was measured. Two polypeptides of p38 MAPK p180T served as the positive control, and UNG2 p31T as the negative control. 10 Assays were also performed in the absence of magnesium or Wip1 and in the presence of okadaic acid. Wip1 phosphatase activity is magnesium-dependent and insensitive to okadaic acid. ( e ) DU145 cells were transfected with BAX or mutated BAX (T172D, T174D, and T186D) expression plasmids. After 48 h, the transfectants were irradiated, and then harvested, and finally assayed for caspase-3 activity. ( f ) DU145 cells were transfected with wild-type or mutated BAX together with wild-type Wip1 or phosphatase-dead (PD) Wip1 (D314A and D105A) expression vectors for 48 h and then irradiated with 10 Gy. After 24 h, the transfectants were assayed for caspase-3 activity. The mean of three experiments is shown in each column; bars in ( d ), ( e ), and ( f ) correspond to the S.D. Asterisks (*) indicate P

    Techniques Used: Recombinant, Derivative Assay, In Vitro, Phosphatase Assay, Expressing, Mutagenesis, Construct, Transfection, Translocation Assay, Fluorescence, Incubation, De-Phosphorylation Assay, Positive Control, Negative Control, Activity Assay, Irradiation

    16) Product Images from "Over-expression of DNA-PKcs in renal cell carcinoma regulates mTORC2 activation, HIF-2α expression and cell proliferation"

    Article Title: Over-expression of DNA-PKcs in renal cell carcinoma regulates mTORC2 activation, HIF-2α expression and cell proliferation

    Journal: Scientific Reports

    doi: 10.1038/srep29415

    DNA-PKcs inhibitors induce proliferation inhibition and apoptosis in RCC cells. 786-0 ( A – E ), A498 ( F ) or primary human RCC cells ( G , H ) as well as the HK-2 human proximal tubule epithelial cells ( I ) were treated with NU-7026 (5 μM), NU-7441 (5 μM), LY-294002 (1 μM) or vehicle control (0.1% DMSO) for applied time, cell proliferation was analyzed by viable cell counting assay ( A , for 786-0 cells), MTT assay ( B , F , G , I ) or clonogenicity assay ( C , for 786-0 cells); Cell apoptosis was tested by the ssDNA ELISA assay ( D , H ) or the caspase-3 activity assay ( E , for 786-0 cells). Experiments in this figure were repeated three times, and similar results were obtained. For each assay, n = 5. * p
    Figure Legend Snippet: DNA-PKcs inhibitors induce proliferation inhibition and apoptosis in RCC cells. 786-0 ( A – E ), A498 ( F ) or primary human RCC cells ( G , H ) as well as the HK-2 human proximal tubule epithelial cells ( I ) were treated with NU-7026 (5 μM), NU-7441 (5 μM), LY-294002 (1 μM) or vehicle control (0.1% DMSO) for applied time, cell proliferation was analyzed by viable cell counting assay ( A , for 786-0 cells), MTT assay ( B , F , G , I ) or clonogenicity assay ( C , for 786-0 cells); Cell apoptosis was tested by the ssDNA ELISA assay ( D , H ) or the caspase-3 activity assay ( E , for 786-0 cells). Experiments in this figure were repeated three times, and similar results were obtained. For each assay, n = 5. * p

    Techniques Used: Inhibition, Cell Counting, MTT Assay, Enzyme-linked Immunosorbent Assay, Caspase-3 Activity Assay

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    Enzyme-linked Immunosorbent Assay:

    Article Title: Effect of Chemotherapeutic Drugs on Caspase-3 Activity, as a Key Biomarker for Apoptosis in Ovarian Tumor Cell Cultured as Monolayer. A Pilot Study
    Article Snippet: After replacing media with caspase assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol and 10 mM DTT), cell lysates were incubated with a caspase-3 colorimetric substrate, acetyl-Asp-Glu-Val-Asp-p-nitroanilide-7-amino-4-methylcoumarin (Ac-DEVD-AMC conjugation); Sigma). .. Reaction mixtures were incubated at 37°C for 15 min to 1h, and the absorbance was measured at 405nm using an ELISA microplate reader (BioTek Instruments).

    Activation Assay:

    Article Title: Selective mGluR1 Antagonist EMQMCM Inhibits the Kainate-Induced Excitotoxicity in Primary Neuronal Cultures and in the Rat Hippocampus
    Article Snippet: Measurement of Caspase-3 Activity For evaluation of apoptosis, caspase-3 activation was measured. .. After replacing the media with Caspase Assay Buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, and 10 mM dithiothreitol), the cell lysates (25 μg per sample) were incubated at 37°C with a colorimetric substrate preferentially cleaved by caspase-3-Ac-DEVD-pNA (N -acetyl-asp-glu-val-asp p -nitro-anilide; 40 μM; Sigma, USA).

    Incubation:

    Article Title: Cordycepin Down-Regulates Multiple Drug Resistant (MDR)/HIF-1α through Regulating AMPK/mTORC1 Signaling in GBC-SD Gallbladder Cancer Cells
    Article Snippet: A total of 30 μg of cytosolic extracts were added to caspase assay buffer (312.5 mm HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) with benzyloxycarbonyl-DEVD-7-amido-4-(trifluoromethyl)coumarin as substrates (Calbiochem, Darmstadt, Germany). .. Release of 7-amido-4-(trifluoromethyl)coumarin (AFC) was detected, after 1 h of incubation at 37 °C with a fluorescence reader (BD), set to an excitation value of 355 nm and emission value of 525 nm.

    Article Title: Small Molecular TRAIL Inducer ONC201 Induces Death in Lung Cancer Cells: A Preclinical Study
    Article Snippet: Caspase activity assay After treatment of cells, cytosolic proteins (30 μg lysates per treatment) were added to the caspase assay buffer (312.5 mM HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) with Ac-DEVD-AFC (10 μg/mL, CalBiochem) as the caspase-3 substrate, or Ac-IETD-AFC (10 μg/mL, CalBiochem) as the caspase-8 substrate. .. After incubation for 1 hour under the dark, the released AFC was measured via a spectrofluorometer with excitation of 400 nm [ ].

    Article Title: Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity
    Article Snippet: .. The reaction was incubated for 0.5 h. For the cross-linking experiment, caspase assay buffer was supplemented with 20 U/ml thrombin (Sigma-Aldrich). .. 1 h later, 2 mM bismaleimidohexane (Thermo Fisher Scientific) was added for 1 h at RT.

    Article Title: The Antipancreatic Cancer Activity of OSI-027, a Potent and Selective Inhibitor of mTORC1 and mTORC2
    Article Snippet: Twenty microgram of cytosolic extracts were added to caspase assay buffer (312.5 mM HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) with Ac-DEVD-AFC (15 μg/mL) (Calbiochem) as the substrate. .. After incubation at 37°C for 1 h, the amount of AFC liberated was measured using a spectrofluorometer (Thermo Labsystems, Helsinki, Finland) with excitation of 380 nm and emission wavelength of 460 nm.

    Article Title: Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against osteosarcoma
    Article Snippet: Twenty μg of cytosolic extracts were added to caspase assay buffer (312.5 mM HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) with Ac-DEVD-AFC (15 μg/ml) (Calbiochem, Darmstadt, Germany) as the substrate. .. After incubation at 37°C for 1 hr, the amount of AFC liberated was measured using a spectrofluorometer (Thermo-Labsystems, Helsinki, Finland) with excitation of 380 nm and emission wavelength of 460 nm.

    Article Title: Effect of Chemotherapeutic Drugs on Caspase-3 Activity, as a Key Biomarker for Apoptosis in Ovarian Tumor Cell Cultured as Monolayer. A Pilot Study
    Article Snippet: .. After replacing media with caspase assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol and 10 mM DTT), cell lysates were incubated with a caspase-3 colorimetric substrate, acetyl-Asp-Glu-Val-Asp-p-nitroanilide-7-amino-4-methylcoumarin (Ac-DEVD-AMC conjugation); Sigma). .. Reaction mixtures were incubated at 37°C for 15 min to 1h, and the absorbance was measured at 405nm using an ELISA microplate reader (BioTek Instruments).

    Article Title: ROS-p53-cyclophilin-D signaling mediates salinomycin-induced glioma cell necrosis
    Article Snippet: Twenty μg of cytosolic extracts were added to caspase assay buffer (312.5 mM HEPES, pH 7.5, 31.25 % sucrose, 0.3125 % CHAPS) with benzyloxycarbonyl-DEVD-7-amido-4-(trifluoromethyl)-coumarin as substrate (Calbiochem, Darmstadt, Germany). .. After 2 h of incubation at 37 °C, the release of 7-amido-4-(trifluoromethyl) coumarin (AFC) was quantified, using a Fluoroskan system set to an excitation value of 355 nm and emission value of 525 nm.

    Article Title: Ceramide Production Mediates Aldosterone-Induced Human Umbilical Vein Endothelial Cell (HUVEC) Damages
    Article Snippet: Thirty μg of cytosolic extracts per sample were added to caspase assay buffer (312.5 mm HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) with benzyloxycarbonyl-DEVD-7-amido-4-(trifluoromethyl)coumarin as the substrate (Calbiochem). .. After 2 hours of incubation at 37°C, the release of 7-amido-4-(trifluoromethyl)coumarin (AFC) was quantified, using a Fluoroskan system (Thermo-Labsystems, Helsinki, Finland) set to an excitation value of 355 nm and emission value of 525 nm as described [ ].

    Article Title: Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings
    Article Snippet: Assay of caspase-3 activity As described [ ], to test caspase-3 activity, 20 μg of cytosolic protein extracts per sample were mixed with the caspase assay buffer [ ] and the caspase-3 substrate Ac-DEVD-AFC (15 μg/mL) (Calbiochem). .. After 1 h incubation at 37°C, the released AFC was measured through a Shimadzu FC 5300 spectrofluorometer with excitation at 400 nm.

    Article Title: Selective mGluR1 Antagonist EMQMCM Inhibits the Kainate-Induced Excitotoxicity in Primary Neuronal Cultures and in the Rat Hippocampus
    Article Snippet: .. After replacing the media with Caspase Assay Buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, and 10 mM dithiothreitol), the cell lysates (25 μg per sample) were incubated at 37°C with a colorimetric substrate preferentially cleaved by caspase-3-Ac-DEVD-pNA (N -acetyl-asp-glu-val-asp p -nitro-anilide; 40 μM; Sigma, USA). ..

    Article Title: The anti-cancer activity of the mTORC1/2 dual inhibitor XL388 in preclinical osteosarcoma models
    Article Snippet: Twenty μg of cytosolic extracts were added to the caspase assay buffer [ , ] with Ac-DEVD-AFC (15 μg/mL) (Calbiochem) as the substrate. .. After incubation, the amount of released AFC was measured using a spectrofluorometer (Thermo-Labsystems, Helsinki, Finland).

    Article Title: Wip1 suppresses apoptotic cell death through direct dephosphorylation of BAX in response to γ-radiation
    Article Snippet: .. Aliquots of protein (50 μ g) were diluted to 50 μ l with Triton X-100 lysis buffer and then incubated for 1 h with an equal volume of 2 × caspase assay buffer (100 mM HEPES, 10% sucrose, 0.1% CHAPS, 1 mM PMSF, 1 mM DTT, 1 × proteinase inhibitor cocktail) containing 50 μ M of fluorogenic (Ac-DEVD-AMC) caspase-3 substrate (235425; Calbiochem, Billerica, MA, USA). .. Cleavage was monitored by excitation at 380 nm and emission at 460 nm using a fluorescence spectrometer (Victor X3, Waltham, MA, USA).

    Activity Assay:

    Article Title: Effect of Chemotherapeutic Drugs on Caspase-3 Activity, as a Key Biomarker for Apoptosis in Ovarian Tumor Cell Cultured as Monolayer. A Pilot Study
    Article Snippet: The comparison of the absorbance of pNA from an apoptotic sample with an uninduced control allows determination of the fold increase in caspase-3 activity. .. After replacing media with caspase assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol and 10 mM DTT), cell lysates were incubated with a caspase-3 colorimetric substrate, acetyl-Asp-Glu-Val-Asp-p-nitroanilide-7-amino-4-methylcoumarin (Ac-DEVD-AMC conjugation); Sigma).

    Article Title: Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings
    Article Snippet: .. Assay of caspase-3 activity As described [ ], to test caspase-3 activity, 20 μg of cytosolic protein extracts per sample were mixed with the caspase assay buffer [ ] and the caspase-3 substrate Ac-DEVD-AFC (15 μg/mL) (Calbiochem). .. After 1 h incubation at 37°C, the released AFC was measured through a Shimadzu FC 5300 spectrofluorometer with excitation at 400 nm.

    Article Title: Selective mGluR1 Antagonist EMQMCM Inhibits the Kainate-Induced Excitotoxicity in Primary Neuronal Cultures and in the Rat Hippocampus
    Article Snippet: Paragraph title: Measurement of Caspase-3 Activity ... After replacing the media with Caspase Assay Buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, and 10 mM dithiothreitol), the cell lysates (25 μg per sample) were incubated at 37°C with a colorimetric substrate preferentially cleaved by caspase-3-Ac-DEVD-pNA (N -acetyl-asp-glu-val-asp p -nitro-anilide; 40 μM; Sigma, USA).

    Article Title: Independent roles of the priming and the triggering of the NLRP3 inflammasome in the heart
    Article Snippet: Paragraph title: 2.6. Measurement of caspase-1 tissue activity ... Briefly, proteins were extracted from frozen hearts and spleens using RIPA buffer (Sigma Aldrich, St Louis, MO, USA) and were diluted in caspase assay buffer (31% sucrose, 3.1% HEPES, and 0.31% CHAPS; Sigma Aldrich).

    Article Title: The preclinical assessment of XL388, a mTOR kinase inhibitor, as a promising anti-renal cell carcinoma agent
    Article Snippet: .. Assay of caspase activity After treatment, 20 μg protein lysates (per treatment) were added to caspase assay buffer [ ] along with the corresponding caspase substrate (Calbiochem, Darmstadt, Germany). ..

    Cell Culture:

    Article Title: Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity
    Article Snippet: Cell culture medium was then replaced with caspase assay buffer (200 mM NaCl, 50 mM Hepes, pH 8.0, 50 mM KCl, and 100 µg/ml digitonin). .. The reaction was incubated for 0.5 h. For the cross-linking experiment, caspase assay buffer was supplemented with 20 U/ml thrombin (Sigma-Aldrich).

    Expressing:

    Article Title: Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity
    Article Snippet: For the substrate cleavage assay, the buffer was supplemented with 10 mM DTT; cell extract from HEK293T expressing pro–IL-1β-V5, pro–IL-18-V5, or pro-GSDMD-V5; and 20 U/ml thrombin (Sigma-Aldrich), where appropriate, to cleave the engineered caspase-1 protein. .. The reaction was incubated for 0.5 h. For the cross-linking experiment, caspase assay buffer was supplemented with 20 U/ml thrombin (Sigma-Aldrich).

    Lysis:

    Article Title: Cordycepin Down-Regulates Multiple Drug Resistant (MDR)/HIF-1α through Regulating AMPK/mTORC1 Signaling in GBC-SD Gallbladder Cancer Cells
    Article Snippet: Caspase-3 Activity Assay After treatment, the cytosolic proteins of GBC-SD cells were extracted in hypotonic cell lysis buffer (25 mm HEPES, pH 7.2, 5 mM MgCl2 , 5 mm EDTA, 5 mM dithiothreitol, 0.05% phenylmethylsulfonyl fluoride). .. A total of 30 μg of cytosolic extracts were added to caspase assay buffer (312.5 mm HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) with benzyloxycarbonyl-DEVD-7-amido-4-(trifluoromethyl)coumarin as substrates (Calbiochem, Darmstadt, Germany).

    Article Title: The Antipancreatic Cancer Activity of OSI-027, a Potent and Selective Inhibitor of mTORC1 and mTORC2
    Article Snippet: After treatment, cytosolic proteins from approximately 2–3×106 cells were extracted in hypotonic cell lysis buffer described in (Zhu et al. , ). .. Twenty microgram of cytosolic extracts were added to caspase assay buffer (312.5 mM HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) with Ac-DEVD-AFC (15 μg/mL) (Calbiochem) as the substrate.

    Article Title: Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against osteosarcoma
    Article Snippet: After treatment, cytosolic proteins from approximately 2–3 × 106 cells were extracted in hypotonic cell lysis buffer described in. .. Twenty μg of cytosolic extracts were added to caspase assay buffer (312.5 mM HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) with Ac-DEVD-AFC (15 μg/ml) (Calbiochem, Darmstadt, Germany) as the substrate.

    Article Title: ROS-p53-cyclophilin-D signaling mediates salinomycin-induced glioma cell necrosis
    Article Snippet: Caspase-3 activity assay The cytosol proteins of approximately 2 × 106 glioma cells were extracted in cell lysis buffer containing 25 mm HEPES, 5 mm MgCl2 , 5 mm EDTA, 5 mm dithiothreitol and 0.05 % phenylmethylsulfonyl fluoride, (pH 7.5). .. Twenty μg of cytosolic extracts were added to caspase assay buffer (312.5 mM HEPES, pH 7.5, 31.25 % sucrose, 0.3125 % CHAPS) with benzyloxycarbonyl-DEVD-7-amido-4-(trifluoromethyl)-coumarin as substrate (Calbiochem, Darmstadt, Germany).

    Article Title: Wip1 suppresses apoptotic cell death through direct dephosphorylation of BAX in response to γ-radiation
    Article Snippet: .. Aliquots of protein (50 μ g) were diluted to 50 μ l with Triton X-100 lysis buffer and then incubated for 1 h with an equal volume of 2 × caspase assay buffer (100 mM HEPES, 10% sucrose, 0.1% CHAPS, 1 mM PMSF, 1 mM DTT, 1 × proteinase inhibitor cocktail) containing 50 μ M of fluorogenic (Ac-DEVD-AMC) caspase-3 substrate (235425; Calbiochem, Billerica, MA, USA). .. Cleavage was monitored by excitation at 380 nm and emission at 460 nm using a fluorescence spectrometer (Victor X3, Waltham, MA, USA).

    Irradiation:

    Article Title: Wip1 suppresses apoptotic cell death through direct dephosphorylation of BAX in response to γ-radiation
    Article Snippet: Caspase-3 activity assay After 24 h of γ -irradiation, DU145 cells were lysed in Triton X-100 lysis buffer (10 mM Tris-HCl, 5 mM EDTA, 320 mM sucrose, 1% Triton X-100, 1 mM PMSF, 2 mM DTT, 1 × proteinase inhibitor cocktail). .. Aliquots of protein (50 μ g) were diluted to 50 μ l with Triton X-100 lysis buffer and then incubated for 1 h with an equal volume of 2 × caspase assay buffer (100 mM HEPES, 10% sucrose, 0.1% CHAPS, 1 mM PMSF, 1 mM DTT, 1 × proteinase inhibitor cocktail) containing 50 μ M of fluorogenic (Ac-DEVD-AMC) caspase-3 substrate (235425; Calbiochem, Billerica, MA, USA).

    Caspase Activity Assay:

    Article Title: Small Molecular TRAIL Inducer ONC201 Induces Death in Lung Cancer Cells: A Preclinical Study
    Article Snippet: .. Caspase activity assay After treatment of cells, cytosolic proteins (30 μg lysates per treatment) were added to the caspase assay buffer (312.5 mM HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) with Ac-DEVD-AFC (10 μg/mL, CalBiochem) as the caspase-3 substrate, or Ac-IETD-AFC (10 μg/mL, CalBiochem) as the caspase-8 substrate. .. After incubation for 1 hour under the dark, the released AFC was measured via a spectrofluorometer with excitation of 400 nm [ ].

    Cleavage Assay:

    Article Title: Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity
    Article Snippet: For the substrate cleavage assay, the buffer was supplemented with 10 mM DTT; cell extract from HEK293T expressing pro–IL-1β-V5, pro–IL-18-V5, or pro-GSDMD-V5; and 20 U/ml thrombin (Sigma-Aldrich), where appropriate, to cleave the engineered caspase-1 protein. .. The reaction was incubated for 0.5 h. For the cross-linking experiment, caspase assay buffer was supplemented with 20 U/ml thrombin (Sigma-Aldrich).

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    Millipore caspase assay buffer
    NVP-BEZ235 activates apoptosis in osteosarcoma cells . MG-63 cells were treated with indicated concentration of NVP-BEZ235 (NVP) for 42 hrs, cell apoptosis was tested by <t>caspase-3</t> activity assay ( A ), Histone-DNA ELISA assay ( B ) and TUNEL staining assay ( C ). The expression of caspase-3 (regular and cleaved) and tubulin (equal loading) was tested by Western blotting ( A , upper panel). MG-63 cells were pretreated with z-VAD-fmk (zvad, 25 μM), z-DVED-fmk (dved, 25 μM) or z-ITED-fmk (ited, 25 μM) for 1 hr, followed by NVP-BEZ235 (200 nM) stimulation, cells were further cultured, before cell growth ( D ) and colony formation ( E ) were tested. Apoptosis in NVP-BEZ235 (200 nM, 42 hrs)-treated U2OS and SaOs-2 cells was analyzed by TUNEL staining assay ( F ). n = 5 for each assay. * P
    Caspase Assay Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase assay buffer/product/Millipore
    Average 97 stars, based on 19 article reviews
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    87
    Millipore caspase lysis buffer
    Delayed hepatocyte injury after induction of ischemia. (A) Evaluation of ALT serum levels and (B) quantification of <t>caspase</t> 3 activity in sham and LPVL-treated rats over a period of up to 48 h following induction of ischemia. (C) Representative microphotographs of HPS staining of the left liver lobe of LPVL-operated rats over a period ranging from 6 to 48 h following ischemia. Liver injury was assessed by the histological evaluation of necrosis. Values are ±SEM of 3–8 different animals. (* P
    Caspase Lysis Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase lysis buffer/product/Millipore
    Average 87 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    caspase lysis buffer - by Bioz Stars, 2020-03
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    NVP-BEZ235 activates apoptosis in osteosarcoma cells . MG-63 cells were treated with indicated concentration of NVP-BEZ235 (NVP) for 42 hrs, cell apoptosis was tested by caspase-3 activity assay ( A ), Histone-DNA ELISA assay ( B ) and TUNEL staining assay ( C ). The expression of caspase-3 (regular and cleaved) and tubulin (equal loading) was tested by Western blotting ( A , upper panel). MG-63 cells were pretreated with z-VAD-fmk (zvad, 25 μM), z-DVED-fmk (dved, 25 μM) or z-ITED-fmk (ited, 25 μM) for 1 hr, followed by NVP-BEZ235 (200 nM) stimulation, cells were further cultured, before cell growth ( D ) and colony formation ( E ) were tested. Apoptosis in NVP-BEZ235 (200 nM, 42 hrs)-treated U2OS and SaOs-2 cells was analyzed by TUNEL staining assay ( F ). n = 5 for each assay. * P

    Journal: Cancer Biology & Therapy

    Article Title: Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against osteosarcoma

    doi: 10.1080/15384047.2015.1017155

    Figure Lengend Snippet: NVP-BEZ235 activates apoptosis in osteosarcoma cells . MG-63 cells were treated with indicated concentration of NVP-BEZ235 (NVP) for 42 hrs, cell apoptosis was tested by caspase-3 activity assay ( A ), Histone-DNA ELISA assay ( B ) and TUNEL staining assay ( C ). The expression of caspase-3 (regular and cleaved) and tubulin (equal loading) was tested by Western blotting ( A , upper panel). MG-63 cells were pretreated with z-VAD-fmk (zvad, 25 μM), z-DVED-fmk (dved, 25 μM) or z-ITED-fmk (ited, 25 μM) for 1 hr, followed by NVP-BEZ235 (200 nM) stimulation, cells were further cultured, before cell growth ( D ) and colony formation ( E ) were tested. Apoptosis in NVP-BEZ235 (200 nM, 42 hrs)-treated U2OS and SaOs-2 cells was analyzed by TUNEL staining assay ( F ). n = 5 for each assay. * P

    Article Snippet: Twenty μg of cytosolic extracts were added to caspase assay buffer (312.5 mM HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) with Ac-DEVD-AFC (15 μg/ml) (Calbiochem, Darmstadt, Germany) as the substrate.

    Techniques: Concentration Assay, Caspase-3 Activity Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Expressing, Western Blot, Cell Culture

    Cordycepin induces GBC-SD cell apoptosis. GBC-SD cells were treated with indicated cordycepin (10–100 μM) for 72 h, cell apoptosis was tested by Annexin V/PI FACS ( A – C ) and Caspase-3 activity assay ( D ); Note that for Annexin V FACS assay, the results of five sets of experiments were quantified ( B , C ); GBC-SD cells were treated with z-VAD-fmk (50 μM) for 1 h, followed by cordycepin (25/100 μM) stimulation, cells were further cultured for 72 h, and cell viability was tested ( E ); Experiments were repeated five times. * Stands for p

    Journal: International Journal of Molecular Sciences

    Article Title: Cordycepin Down-Regulates Multiple Drug Resistant (MDR)/HIF-1α through Regulating AMPK/mTORC1 Signaling in GBC-SD Gallbladder Cancer Cells

    doi: 10.3390/ijms150712778

    Figure Lengend Snippet: Cordycepin induces GBC-SD cell apoptosis. GBC-SD cells were treated with indicated cordycepin (10–100 μM) for 72 h, cell apoptosis was tested by Annexin V/PI FACS ( A – C ) and Caspase-3 activity assay ( D ); Note that for Annexin V FACS assay, the results of five sets of experiments were quantified ( B , C ); GBC-SD cells were treated with z-VAD-fmk (50 μM) for 1 h, followed by cordycepin (25/100 μM) stimulation, cells were further cultured for 72 h, and cell viability was tested ( E ); Experiments were repeated five times. * Stands for p

    Article Snippet: A total of 30 μg of cytosolic extracts were added to caspase assay buffer (312.5 mm HEPES, pH 7.5, 31.25% sucrose, 0.3125% CHAPS) with benzyloxycarbonyl-DEVD-7-amido-4-(trifluoromethyl)coumarin as substrates (Calbiochem, Darmstadt, Germany).

    Techniques: FACS, Caspase-3 Activity Assay, Cell Culture

    ODE activates apoptosis in CRC cells A panel of established CRC cell lines (HCT-116, Lovo, HT-29 and DLD-1) and three primary human CRC cell lines were treated with or without ODE at applied concentrations, cells were further cultured, cell apoptosis was analyzed by listed assay A - D , G and H . HCT-116 cells were pretreated with Ac-DEVD-CHO (“DVED”), Ac-LEHD-CHO (“LEHD”) or Ac-VAD-CHO (“VAD”) (40 μM each) for 1 h, following by ODE (50 μg/mL) treatment, cell viability E . and cell death F . were tested. “DMSO” stands for 0.1% DMSO. Cleaved-PARP/cleaved-caspase-3 expression (vs. Tubulin) was quantified. Data in this figure were repeated four times, and similar results were obtained. * p

    Journal: Oncotarget

    Article Title: Aqueous Oldenlandia diffusa extracts inhibits colorectal cancer cells via activating AMP-activated protein kinase signalings

    doi: 10.18632/oncotarget.9969

    Figure Lengend Snippet: ODE activates apoptosis in CRC cells A panel of established CRC cell lines (HCT-116, Lovo, HT-29 and DLD-1) and three primary human CRC cell lines were treated with or without ODE at applied concentrations, cells were further cultured, cell apoptosis was analyzed by listed assay A - D , G and H . HCT-116 cells were pretreated with Ac-DEVD-CHO (“DVED”), Ac-LEHD-CHO (“LEHD”) or Ac-VAD-CHO (“VAD”) (40 μM each) for 1 h, following by ODE (50 μg/mL) treatment, cell viability E . and cell death F . were tested. “DMSO” stands for 0.1% DMSO. Cleaved-PARP/cleaved-caspase-3 expression (vs. Tubulin) was quantified. Data in this figure were repeated four times, and similar results were obtained. * p

    Article Snippet: Assay of caspase-3 activity As described [ ], to test caspase-3 activity, 20 μg of cytosolic protein extracts per sample were mixed with the caspase assay buffer [ ] and the caspase-3 substrate Ac-DEVD-AFC (15 μg/mL) (Calbiochem).

    Techniques: Cell Culture, Expressing

    Delayed hepatocyte injury after induction of ischemia. (A) Evaluation of ALT serum levels and (B) quantification of caspase 3 activity in sham and LPVL-treated rats over a period of up to 48 h following induction of ischemia. (C) Representative microphotographs of HPS staining of the left liver lobe of LPVL-operated rats over a period ranging from 6 to 48 h following ischemia. Liver injury was assessed by the histological evaluation of necrosis. Values are ±SEM of 3–8 different animals. (* P

    Journal: PLoS ONE

    Article Title: Upregulation of Krebs cycle and anaerobic glycolysis activity early after onset of liver ischemia

    doi: 10.1371/journal.pone.0199177

    Figure Lengend Snippet: Delayed hepatocyte injury after induction of ischemia. (A) Evaluation of ALT serum levels and (B) quantification of caspase 3 activity in sham and LPVL-treated rats over a period of up to 48 h following induction of ischemia. (C) Representative microphotographs of HPS staining of the left liver lobe of LPVL-operated rats over a period ranging from 6 to 48 h following ischemia. Liver injury was assessed by the histological evaluation of necrosis. Values are ±SEM of 3–8 different animals. (* P

    Article Snippet: Caspase 3 activity As described previously [ ], sections of frozen tissue were lysed in ice-cold caspase lysis buffer (10mmol/L HEPES [pH 7.4], 5mmol/L MgCl2, 42mmol/L KCl, 0.1mmol/L EDTA, 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-2-hydroxy-1-propanesulonate (Calbiochem, San Diego, CA, USA), 0.1% Triton X-100, 1mmol/L dithiothreitol (DTT), 1mmol/L phenylmethyl-sulfonyl fluoride, 10μg/mL leupeptin, 10μg/mL aprotinin, 10μg/mL soybean trypsin inhibitor, and 100μmol/L benzamidine).

    Techniques: Activity Assay, Staining