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  • 95
    New England Biolabs t7 express lysy
    T7 Express Lysy, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t7 express lysy/product/New England Biolabs
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    97
    R&D Systems caspase 3
    Changes in the small intestinal epithelium of acute and chronic TNF-mediated injury mouse models. a Schematic of experimental treatment and sampling timeline for acute and chronic TNF-mediated inflammatory injury. b Morphology of duodenal sections illustrating epithelial disruption 1–4 h following a high-dose pulse of TNF (acute model) with concomitant BrdU administration (brown staining), counterstained with Haematoxylin (blue/purple). Arrows indicate the hollow villus tips following stromal retraction induced by TNF and the constriction of the epithelium over the stroma preceding the shedding of the tip, which is re-epithelised at 4 h post-TNF. The epithelium in healthy and chronic inflammation models exhibits standard morphological appearance. Progression of BrdU-labelled cells on the CVEU over time was used to quantify cell dynamics in later analyses. c Images of TUNEL and <t>cleaved-Caspase-3</t> (CC3) labelled duodenum sections illustrating labelling similarity and differences in cell death intensity along the CVEU of healthy and inflammation mouse models. d Representative images and quantification of cells staining positive for goblet cell mucin (MUC2) in small intestine of control and acute inflammation mouse model at 1, 1.5, and 12 h post-TNF delivery. e Plot symbols show the decrease and recovery of the CVEU length (average number of cells ± standard deviation) in duodenum and ileum over time following the administration of one high-dose pulse of TNF (acute inflammation). Continuous blue and red bands show the average ± standard deviation of the CVEU length in control conditions and the chronic inflammation model, respectively
    Caspase 3, supplied by R&D Systems, used in various techniques. Bioz Stars score: 97/100, based on 1453 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    R&D Systems recombinant caspase 3
    NS3694 inhibits TNF-induced effector caspase activation and apoptosis in <t>MCF-casp3</t> cells. MCF-casp3 cells were left untreated (UN) or pretreated with the indicated concentrations of DEVD-CHO or zVAD-fmk for 1 h before the indicated concentrations of TNF were added. NS3694 was added at 10 to 100 μM at the same time as TNF. (A) The survival of the cells was analyzed by the MTT assay after 48 h of TNF treatment. The means ± standard deviations (error bars) of three independent experiments are shown. (B) After 17 h of treatment, the cells were lysed, and the DEVDase activity in lysates was assessed by spectrofluorometric quantification of DEVD-AFC cleavage and presented as picomoles per minute. The means ± standard deviations (error bars) for three samples in a representative experiment are shown. The experiments were repeated several times with similar results.
    Recombinant Caspase 3, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Changes in the small intestinal epithelium of acute and chronic TNF-mediated injury mouse models. a Schematic of experimental treatment and sampling timeline for acute and chronic TNF-mediated inflammatory injury. b Morphology of duodenal sections illustrating epithelial disruption 1–4 h following a high-dose pulse of TNF (acute model) with concomitant BrdU administration (brown staining), counterstained with Haematoxylin (blue/purple). Arrows indicate the hollow villus tips following stromal retraction induced by TNF and the constriction of the epithelium over the stroma preceding the shedding of the tip, which is re-epithelised at 4 h post-TNF. The epithelium in healthy and chronic inflammation models exhibits standard morphological appearance. Progression of BrdU-labelled cells on the CVEU over time was used to quantify cell dynamics in later analyses. c Images of TUNEL and cleaved-Caspase-3 (CC3) labelled duodenum sections illustrating labelling similarity and differences in cell death intensity along the CVEU of healthy and inflammation mouse models. d Representative images and quantification of cells staining positive for goblet cell mucin (MUC2) in small intestine of control and acute inflammation mouse model at 1, 1.5, and 12 h post-TNF delivery. e Plot symbols show the decrease and recovery of the CVEU length (average number of cells ± standard deviation) in duodenum and ileum over time following the administration of one high-dose pulse of TNF (acute inflammation). Continuous blue and red bands show the average ± standard deviation of the CVEU length in control conditions and the chronic inflammation model, respectively

    Journal: Cell Death & Disease

    Article Title: Elevated apoptosis impairs epithelial cell turnover and shortens villi in TNF-driven intestinal inflammation

    doi: 10.1038/s41419-018-1275-5

    Figure Lengend Snippet: Changes in the small intestinal epithelium of acute and chronic TNF-mediated injury mouse models. a Schematic of experimental treatment and sampling timeline for acute and chronic TNF-mediated inflammatory injury. b Morphology of duodenal sections illustrating epithelial disruption 1–4 h following a high-dose pulse of TNF (acute model) with concomitant BrdU administration (brown staining), counterstained with Haematoxylin (blue/purple). Arrows indicate the hollow villus tips following stromal retraction induced by TNF and the constriction of the epithelium over the stroma preceding the shedding of the tip, which is re-epithelised at 4 h post-TNF. The epithelium in healthy and chronic inflammation models exhibits standard morphological appearance. Progression of BrdU-labelled cells on the CVEU over time was used to quantify cell dynamics in later analyses. c Images of TUNEL and cleaved-Caspase-3 (CC3) labelled duodenum sections illustrating labelling similarity and differences in cell death intensity along the CVEU of healthy and inflammation mouse models. d Representative images and quantification of cells staining positive for goblet cell mucin (MUC2) in small intestine of control and acute inflammation mouse model at 1, 1.5, and 12 h post-TNF delivery. e Plot symbols show the decrease and recovery of the CVEU length (average number of cells ± standard deviation) in duodenum and ileum over time following the administration of one high-dose pulse of TNF (acute inflammation). Continuous blue and red bands show the average ± standard deviation of the CVEU length in control conditions and the chronic inflammation model, respectively

    Article Snippet: Labelled cell counts for BrdU, Ki-67, caspase 3 and TUNEL were obtained following the format described in ref. .

    Techniques: Sampling, Staining, TUNEL Assay, Standard Deviation

    Immunohistochemical localization of CASP3 and TNF proteins in CL of the domestic cat. Active CASP3 was localized in luteal cells at the stages of: CL formation (A), development/maintenance (B), early regression (C) and late regression (D). The protein expression was consistently present throughout the CL life span, except for CA (E). TNF protein expression was identified in non-steroidogenic cells, presumably macrophages, but not in luteal cells (F). Arrows indicate positive staining. NC: negative control. Scale bar– 50 μm.

    Journal: PLoS ONE

    Article Title: Apoptosis-Related Factors in the Luteal Phase of the Domestic Cat and Their Involvement in the Persistence of Corpora Lutea in Lynx

    doi: 10.1371/journal.pone.0143414

    Figure Lengend Snippet: Immunohistochemical localization of CASP3 and TNF proteins in CL of the domestic cat. Active CASP3 was localized in luteal cells at the stages of: CL formation (A), development/maintenance (B), early regression (C) and late regression (D). The protein expression was consistently present throughout the CL life span, except for CA (E). TNF protein expression was identified in non-steroidogenic cells, presumably macrophages, but not in luteal cells (F). Arrows indicate positive staining. NC: negative control. Scale bar– 50 μm.

    Article Snippet: The localization of CASP3 protein in luteal tissue was assessed by an affinity-purified rabbit anti-human/mouse CASP3 reactive antibody (Cat. AF 835; R & D Systems, Wiesbaden, Germany), used in a previous study on the domestic cat [ ].

    Techniques: Immunohistochemistry, Expressing, Staining, Negative Control

    Immunohistochemical localization of CASP3 and TNF proteins in CL of Iberian and Eurasian lynx. A. Negative staining for active CASP3 in lynx CL tissue. B. frCL of Iberian lynx; strong staining for TNF protein in transforming cells (arrows). C. perCL of Iberian lynx; staining for TNF protein in non-steroidogenic cells, presumably macrophages (arrows). D. perCL of Eurasian lynx; staining for TNF protein in non-steroidogenic cells, presumably macrophages (arrows). Staining of droplets within the cells is unspecific. NC: negative control. Scale bar– 50 μm.

    Journal: PLoS ONE

    Article Title: Apoptosis-Related Factors in the Luteal Phase of the Domestic Cat and Their Involvement in the Persistence of Corpora Lutea in Lynx

    doi: 10.1371/journal.pone.0143414

    Figure Lengend Snippet: Immunohistochemical localization of CASP3 and TNF proteins in CL of Iberian and Eurasian lynx. A. Negative staining for active CASP3 in lynx CL tissue. B. frCL of Iberian lynx; strong staining for TNF protein in transforming cells (arrows). C. perCL of Iberian lynx; staining for TNF protein in non-steroidogenic cells, presumably macrophages (arrows). D. perCL of Eurasian lynx; staining for TNF protein in non-steroidogenic cells, presumably macrophages (arrows). Staining of droplets within the cells is unspecific. NC: negative control. Scale bar– 50 μm.

    Article Snippet: The localization of CASP3 protein in luteal tissue was assessed by an affinity-purified rabbit anti-human/mouse CASP3 reactive antibody (Cat. AF 835; R & D Systems, Wiesbaden, Germany), used in a previous study on the domestic cat [ ].

    Techniques: Immunohistochemistry, Negative Staining, Staining, Negative Control

    Sulforaphane induces Cx43 and inhibits CSC characteristics in primary CSCs (A) Staining of patient-derived frozen tissue from a ductal adenocarcinoma (Primary Tumor 22) and it´s derived xenograft from passage 3 (Xenograft P3) with Trichrome, c-Met or Cx43 phosphorylated at Ser 368 (Abcam), followed by microscopical evaluation under 400× magnification. The scale bar indicates 50 μm. (B) Representative picture of an anchorage-independent growing spheroidal culture established from a mouse xenograft derived from the primary patient tumor 22. The percentage of expression of the CSC markers c-Met and CD133 was determined as described in part C and is indicated. (C) One week after in vitro spheroidal culture, cells derived from pancreatic ductal adenocarcinoma 22 and 30 were left untreated or were treated with sulforaphane (10 μM). Twenty-four hours later, the cells were cytospinned to glass slides, and the expression of Cx43 phosphorylated at Ser 368 (Abcam), c-Met, CD133, E-cadherin and the cleaved, active fragment of Caspase-3 was examined by immunohistochemistry. The number of positive cells was quantified in 10 vision fields under 400× magnification and the means ± SD are shown in the diagrams. **p

    Journal: Oncotarget

    Article Title: Sulforaphane counteracts aggressiveness of pancreatic cancer driven by dysregulated Cx43-mediated gap junctional intercellular communication

    doi:

    Figure Lengend Snippet: Sulforaphane induces Cx43 and inhibits CSC characteristics in primary CSCs (A) Staining of patient-derived frozen tissue from a ductal adenocarcinoma (Primary Tumor 22) and it´s derived xenograft from passage 3 (Xenograft P3) with Trichrome, c-Met or Cx43 phosphorylated at Ser 368 (Abcam), followed by microscopical evaluation under 400× magnification. The scale bar indicates 50 μm. (B) Representative picture of an anchorage-independent growing spheroidal culture established from a mouse xenograft derived from the primary patient tumor 22. The percentage of expression of the CSC markers c-Met and CD133 was determined as described in part C and is indicated. (C) One week after in vitro spheroidal culture, cells derived from pancreatic ductal adenocarcinoma 22 and 30 were left untreated or were treated with sulforaphane (10 μM). Twenty-four hours later, the cells were cytospinned to glass slides, and the expression of Cx43 phosphorylated at Ser 368 (Abcam), c-Met, CD133, E-cadherin and the cleaved, active fragment of Caspase-3 was examined by immunohistochemistry. The number of positive cells was quantified in 10 vision fields under 400× magnification and the means ± SD are shown in the diagrams. **p

    Article Snippet: Primary Antibodies Rabbit polyclonal antibodies (pAbs) were used against Cx32, Cx26 (Invitrogen, Camarillo, California; USA), c-Met, total Cx43 (#3512, Cell Signaling Technology, Boston, MA, USA), Cx43 phosphorylated at Ser 368 (#ab47368; Abcam, Cambridge, UK), Cx45 phosphorylated at Ser 279/282 (Santa Cruz Biotechnology, Inc. Heidelberg, Germany), Cx45, CD44 (GeneTex, Irvine, California, USA), acetyl-Histone H3 and acetyl-Histone H4 (Merck Millipore, Darmstadt, Germany), E-cadherin (Cell Signaling), Ki67 (Thermo Scientific, Rockford, IL, USA) and the cleaved fragment of activated human caspase-3 (R & D Systems, Abingdon, UK).

    Techniques: Staining, Derivative Assay, Expressing, In Vitro, Immunohistochemistry

    Caspase 3 is cleaved and activated when Akt signaling is suppressed. (A) Human VSMC were infected with adenovirus vector expressing dominant-negative Akt (dnAkt) (MOI = 100) after preinfection with adenovirus vectors expressing β-Gal or dominant-negative caspase 9 (dnCasp-9) at an MOI of 100. Control (mock infected) and Adeno-β Gal-infected cultures were incubated in parallel with the Adeno-dnAkt-infected cultures. Cell lysates were prepared 24 h after the second infection. Immunoblot (IB) analysis with anti-caspase 3 antibody was performed on anti-caspase 3-immunoprecipitated (IP) material. (B) VSMC were preinfected with an adenoviral vector expressing CrmA 12 h prior to infection with Adeno-dnAkt. Cell lysates were prepared 24 or 48 h after infection with Adeno-dnAkt. (C) VSMC were infected with adenovirus vector expressing dnAkt or β-Gal (MOI of 50 or 100) in the presence or absence of Z-DEVD (20 μM). Control cultures were incubated in parallel but did not receive treatment with adenovirus or Z-DEVD. Anoikis was used as a positive control in which cells were placed in suspension cultures for 15 h. Cell lysates were extracted 15 h after infection, and immunoblot analysis with anti-caspase 3 antibody was performed on anti-caspase 3-immunoprecipitated material. (D) Caspase activity was determined using a caspase 3 colorimetric assay which involved the incubation of cell lysates with caspase 3-specific peptide and quantitation with a scanning multiwell spectrophotometer. Values are presented as means ± SEM ( n = 4).

    Journal: Molecular and Cellular Biology

    Article Title: Suppression of Akt Signaling Induces Fas Ligand Expression: Involvement of Caspase and Jun Kinase Activation in Akt-Mediated Fas Ligand Regulation

    doi: 10.1128/MCB.22.2.680-691.2002

    Figure Lengend Snippet: Caspase 3 is cleaved and activated when Akt signaling is suppressed. (A) Human VSMC were infected with adenovirus vector expressing dominant-negative Akt (dnAkt) (MOI = 100) after preinfection with adenovirus vectors expressing β-Gal or dominant-negative caspase 9 (dnCasp-9) at an MOI of 100. Control (mock infected) and Adeno-β Gal-infected cultures were incubated in parallel with the Adeno-dnAkt-infected cultures. Cell lysates were prepared 24 h after the second infection. Immunoblot (IB) analysis with anti-caspase 3 antibody was performed on anti-caspase 3-immunoprecipitated (IP) material. (B) VSMC were preinfected with an adenoviral vector expressing CrmA 12 h prior to infection with Adeno-dnAkt. Cell lysates were prepared 24 or 48 h after infection with Adeno-dnAkt. (C) VSMC were infected with adenovirus vector expressing dnAkt or β-Gal (MOI of 50 or 100) in the presence or absence of Z-DEVD (20 μM). Control cultures were incubated in parallel but did not receive treatment with adenovirus or Z-DEVD. Anoikis was used as a positive control in which cells were placed in suspension cultures for 15 h. Cell lysates were extracted 15 h after infection, and immunoblot analysis with anti-caspase 3 antibody was performed on anti-caspase 3-immunoprecipitated material. (D) Caspase activity was determined using a caspase 3 colorimetric assay which involved the incubation of cell lysates with caspase 3-specific peptide and quantitation with a scanning multiwell spectrophotometer. Values are presented as means ± SEM ( n = 4).

    Article Snippet: Caspase 3 activity was measured using the caspase 3 colorimetric assay kit according to the directions of the manufacturer (R & D Systems).

    Techniques: Infection, Plasmid Preparation, Expressing, Dominant Negative Mutation, Incubation, Immunoprecipitation, Positive Control, Activity Assay, Colorimetric Assay, Quantitation Assay, Spectrophotometry

    Ghrelin therapy decreases caspase-3 after CI. Animals were irradiated alone or followed by wounding. Blood on different time points were collected for measuring KC concentrations in serum (N = 6 per group). *p

    Journal: Cell & Bioscience

    Article Title: Ghrelin therapy mitigates bone marrow injury and splenocytopenia by sustaining circulating G-CSF and KC increases after irradiation combined with wound

    doi: 10.1186/s13578-018-0225-3

    Figure Lengend Snippet: Ghrelin therapy decreases caspase-3 after CI. Animals were irradiated alone or followed by wounding. Blood on different time points were collected for measuring KC concentrations in serum (N = 6 per group). *p

    Article Snippet: Activated caspase-3 protein levels were measured using Quantikine ELISA kit according to the manufacturer’s protocol (R & D SYSTEM, Minneapolis, MN).

    Techniques: Irradiation

    The role of AMPK in the effects of cilostazol on ethanol-induced apoptosis of hepatocytes. (A) Cells were treated with cilostazol (0 ~ 100 μM) in the presence or absence of compound C (10 μM) for 10 min. Phosphorylated- and total-AMPK and ACC were detected by Western blotting. (B—D) Cells were treated with ethanol (100 and 200 mM) in the presence of AICAR (100 μM), cilostazol (100 μM), compound C (10 μM) or compound C (10 μM) plus cilostazol (100 μM) for 24 h. Cleaved caspase-3 (C-caspase-3) or cleaved PARP (C-PARP) were detected by Western blotting (B, C). The caspase-3 activity was measured (D). (E) Cells were treated with AMPK siRNA or scramble siRNA for 48 h and then treated with cilostazol (100 μM) for 10 min. Phosphorylated- and total-AMPK and ACC were detected by Western blotting. (F, G) Cells were treated with AMPK siRNA or scramble siRNA for 24 h and then stimulated with ethanol (100 mM) in the presence or absence of cilostazol (100 μM) for 24 h. Caspase-3 activity (F) and cell viability (G) were measured. Representative microscopic images from three independent experiments were presented (magnification, x 400). The blots are representative of three independent experiments. Data represented as fold increases is mean±S.E.M. of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Cilostazol protects hepatocytes against alcohol-induced apoptosis via activation of AMPK pathway

    doi: 10.1371/journal.pone.0211415

    Figure Lengend Snippet: The role of AMPK in the effects of cilostazol on ethanol-induced apoptosis of hepatocytes. (A) Cells were treated with cilostazol (0 ~ 100 μM) in the presence or absence of compound C (10 μM) for 10 min. Phosphorylated- and total-AMPK and ACC were detected by Western blotting. (B—D) Cells were treated with ethanol (100 and 200 mM) in the presence of AICAR (100 μM), cilostazol (100 μM), compound C (10 μM) or compound C (10 μM) plus cilostazol (100 μM) for 24 h. Cleaved caspase-3 (C-caspase-3) or cleaved PARP (C-PARP) were detected by Western blotting (B, C). The caspase-3 activity was measured (D). (E) Cells were treated with AMPK siRNA or scramble siRNA for 48 h and then treated with cilostazol (100 μM) for 10 min. Phosphorylated- and total-AMPK and ACC were detected by Western blotting. (F, G) Cells were treated with AMPK siRNA or scramble siRNA for 24 h and then stimulated with ethanol (100 mM) in the presence or absence of cilostazol (100 μM) for 24 h. Caspase-3 activity (F) and cell viability (G) were measured. Representative microscopic images from three independent experiments were presented (magnification, x 400). The blots are representative of three independent experiments. Data represented as fold increases is mean±S.E.M. of three independent experiments. * P

    Article Snippet: A recombinant caspase-3 enzyme (R & D Systems, Inc., Minneapolis, MN) was used for generating a standard and caspase-3 activity was calculated as ng/mg protein.

    Techniques: Western Blot, Activity Assay

    The regulation of AMPK by LKB1 and CaMKK2 in cilostazol treated hepatocytes. (A) Cells were treated with LKB1- and CaMKK2- siRNA or scramble siRNA for 48 h and then treated with cilostazol (100 μM) for 10 min. Phosphorylated- and total-LKB1, CaMKK2, AMPK and ACC were detected by Western blotting. (B, C) Cells were treated with LKB1- and CaMKK2- siRNA or scramble siRNA for 24 h and then stimulated with ethanol (100 mM) in the presence or absence of cilostazol (100 μM) for 24 h. Caspase-3 activity (B) and cell viability (C) were measured. (D) Cells were treated with cilostazol (100 μM) for 10 min in the presence or absence of KT5720 (1 μM), SQ22536 (400 μM) or STO-609 (5 μM). Phosphorylated- and total- AMPK and ACC were detected by Western blotting. (E) Cells were treated with ethanol (100 mM) in the presence or absence of cilostazol (100 μM), STO-609 (5 μM) or KT5720 (1 μM) for 24 h. Then, cell viability was measured by MTS assay. The blots are representative of three independent experiments. Data represented as fold increases is mean±S.E.M. of three independent experiments. *** P

    Journal: PLoS ONE

    Article Title: Cilostazol protects hepatocytes against alcohol-induced apoptosis via activation of AMPK pathway

    doi: 10.1371/journal.pone.0211415

    Figure Lengend Snippet: The regulation of AMPK by LKB1 and CaMKK2 in cilostazol treated hepatocytes. (A) Cells were treated with LKB1- and CaMKK2- siRNA or scramble siRNA for 48 h and then treated with cilostazol (100 μM) for 10 min. Phosphorylated- and total-LKB1, CaMKK2, AMPK and ACC were detected by Western blotting. (B, C) Cells were treated with LKB1- and CaMKK2- siRNA or scramble siRNA for 24 h and then stimulated with ethanol (100 mM) in the presence or absence of cilostazol (100 μM) for 24 h. Caspase-3 activity (B) and cell viability (C) were measured. (D) Cells were treated with cilostazol (100 μM) for 10 min in the presence or absence of KT5720 (1 μM), SQ22536 (400 μM) or STO-609 (5 μM). Phosphorylated- and total- AMPK and ACC were detected by Western blotting. (E) Cells were treated with ethanol (100 mM) in the presence or absence of cilostazol (100 μM), STO-609 (5 μM) or KT5720 (1 μM) for 24 h. Then, cell viability was measured by MTS assay. The blots are representative of three independent experiments. Data represented as fold increases is mean±S.E.M. of three independent experiments. *** P

    Article Snippet: A recombinant caspase-3 enzyme (R & D Systems, Inc., Minneapolis, MN) was used for generating a standard and caspase-3 activity was calculated as ng/mg protein.

    Techniques: Western Blot, Activity Assay, MTS Assay

    The role of cAMP in the effects of cilostazol on ethanol-induced apoptosis of hepatocytes. (A) Cells were treated with cilostazol (100 μM), forskolin (10 μM) for 30 min in the presence or absence of SQ22536 (400 μM). Intracellular cAMP concentration was measured. (B) Cells were treated with ethanol (100 mM) for 24 h in the presence of forskolin (1 ~ 10 μM), cilostazol (100 μM), or SQ22536 (400 μM). Caspase-3 activity was measured. Data represented as fold increases are mean±S.E.M. of three independent experiments. ** P

    Journal: PLoS ONE

    Article Title: Cilostazol protects hepatocytes against alcohol-induced apoptosis via activation of AMPK pathway

    doi: 10.1371/journal.pone.0211415

    Figure Lengend Snippet: The role of cAMP in the effects of cilostazol on ethanol-induced apoptosis of hepatocytes. (A) Cells were treated with cilostazol (100 μM), forskolin (10 μM) for 30 min in the presence or absence of SQ22536 (400 μM). Intracellular cAMP concentration was measured. (B) Cells were treated with ethanol (100 mM) for 24 h in the presence of forskolin (1 ~ 10 μM), cilostazol (100 μM), or SQ22536 (400 μM). Caspase-3 activity was measured. Data represented as fold increases are mean±S.E.M. of three independent experiments. ** P

    Article Snippet: A recombinant caspase-3 enzyme (R & D Systems, Inc., Minneapolis, MN) was used for generating a standard and caspase-3 activity was calculated as ng/mg protein.

    Techniques: Concentration Assay, Activity Assay

    Effects of cilostazol on ethanol-induced apoptosis of hepatocytes. (A, B) Cells were treated with 100 mM of ethanol for 24 h in the presence or absence of different concentration of cilostazol (0 ~ 100 μM). Cell viability (A) and caspase-3 activity (B) were measured by MTS assay and activity assay as described in “methods”. (C—E) Cells were treated with 100 mM or 200 mM ethanol for 24 h in the presence or absence of cilostazol (100 μM) alone or together with compound C (10 μM). The cleaved caspase-3 (C-caspase-3) and cleaved PARP (C-PARP) were detected by Western blotting. The blots are representative of four independent experiments. Protein levels were normalized to GAPDH. The relative band intensities are presented (C). Nuclear morphology was monitored by fluorescence microscopy after staining cells with Hoechst 33342. Arrows indicate nuclear condensation or nuclear fragmentation (D). TUNEL positive cells were monitored by fluorescence microscopy after staining cells with propidium iodide (red) and TUNEL (green) (E). Representative microscopic images from three independent experiments were presented (magnification, x 400). Data represented as fold increases are mean±S.E.M. of three independent experiments. * P

    Journal: PLoS ONE

    Article Title: Cilostazol protects hepatocytes against alcohol-induced apoptosis via activation of AMPK pathway

    doi: 10.1371/journal.pone.0211415

    Figure Lengend Snippet: Effects of cilostazol on ethanol-induced apoptosis of hepatocytes. (A, B) Cells were treated with 100 mM of ethanol for 24 h in the presence or absence of different concentration of cilostazol (0 ~ 100 μM). Cell viability (A) and caspase-3 activity (B) were measured by MTS assay and activity assay as described in “methods”. (C—E) Cells were treated with 100 mM or 200 mM ethanol for 24 h in the presence or absence of cilostazol (100 μM) alone or together with compound C (10 μM). The cleaved caspase-3 (C-caspase-3) and cleaved PARP (C-PARP) were detected by Western blotting. The blots are representative of four independent experiments. Protein levels were normalized to GAPDH. The relative band intensities are presented (C). Nuclear morphology was monitored by fluorescence microscopy after staining cells with Hoechst 33342. Arrows indicate nuclear condensation or nuclear fragmentation (D). TUNEL positive cells were monitored by fluorescence microscopy after staining cells with propidium iodide (red) and TUNEL (green) (E). Representative microscopic images from three independent experiments were presented (magnification, x 400). Data represented as fold increases are mean±S.E.M. of three independent experiments. * P

    Article Snippet: A recombinant caspase-3 enzyme (R & D Systems, Inc., Minneapolis, MN) was used for generating a standard and caspase-3 activity was calculated as ng/mg protein.

    Techniques: Concentration Assay, Activity Assay, MTS Assay, Western Blot, Fluorescence, Microscopy, Staining, TUNEL Assay

    Effects of cilostazol on apoptosis and AMPK signaling in liver from binge alcohol treated mice. (A) After ethanol binge drinking (5 g/kg body weight) by oral gavage, caspase-3 activity was measured at different times. (B—D) Mice were subjected to intraperitoneal injection of cilostazol (10 mg/kg/day) for 4 days following a single dose of ethanol (5 g/kg) intake. Liver samples were taken at 6 h after ethanol intake. (C) Liver sections were stained with H E (a-c) or anti-cleaved caspase-3 antibody (d-f). Cleaved caspase-3 positive cells showed cytoplasmic tan staining (magnification, x 400). Caspase-3 activity and AMPK signaling were measured by activity assay (B) and Western blotting (D), respectively. Three representative lanes of five different samples are shown in each group. Data represented as fold increases (A, B) or % of relative band intensity (D) are mean±S.E.M. (n = 5 mice per group). * P

    Journal: PLoS ONE

    Article Title: Cilostazol protects hepatocytes against alcohol-induced apoptosis via activation of AMPK pathway

    doi: 10.1371/journal.pone.0211415

    Figure Lengend Snippet: Effects of cilostazol on apoptosis and AMPK signaling in liver from binge alcohol treated mice. (A) After ethanol binge drinking (5 g/kg body weight) by oral gavage, caspase-3 activity was measured at different times. (B—D) Mice were subjected to intraperitoneal injection of cilostazol (10 mg/kg/day) for 4 days following a single dose of ethanol (5 g/kg) intake. Liver samples were taken at 6 h after ethanol intake. (C) Liver sections were stained with H E (a-c) or anti-cleaved caspase-3 antibody (d-f). Cleaved caspase-3 positive cells showed cytoplasmic tan staining (magnification, x 400). Caspase-3 activity and AMPK signaling were measured by activity assay (B) and Western blotting (D), respectively. Three representative lanes of five different samples are shown in each group. Data represented as fold increases (A, B) or % of relative band intensity (D) are mean±S.E.M. (n = 5 mice per group). * P

    Article Snippet: A recombinant caspase-3 enzyme (R & D Systems, Inc., Minneapolis, MN) was used for generating a standard and caspase-3 activity was calculated as ng/mg protein.

    Techniques: Mouse Assay, Activity Assay, Injection, Staining, Western Blot

    Increased active CASP3 is greater in the lungs of γδ −/− mice exposed to acrolein. The accumulation of apoptotic cells in airway epithelial cells of mice was assessed by immunohistochemistry on paraffin-embedded sections

    Journal:

    Article Title: Nonredundant Functions of ?? and ?? T Cells in Acrolein-Induced Pulmonary Pathology

    doi: 10.1093/toxsci/kfn106

    Figure Lengend Snippet: Increased active CASP3 is greater in the lungs of γδ −/− mice exposed to acrolein. The accumulation of apoptotic cells in airway epithelial cells of mice was assessed by immunohistochemistry on paraffin-embedded sections

    Article Snippet: Activate CASPASE 3 (CASP3) protein was detected with a rabbit polyclonal antibody (R & D Systems, Minneapolis, MN) in paraffin sections (5 μm) of mouse airways as previously described ( ).

    Techniques: Mouse Assay, Immunohistochemistry

    Tom20-induced translocation of Bax to mitochondria contributes to pyroptosis. Melanoma A375 cells were treated with CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 for 6 h to detect the translocation of Bax to mitochondria, cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. To detect the effects of the Tom20 point mutants Tom20 C13S and Tom20 C21S , Tom20 was knocked down in A375 cells, and Tom20 WT or its point mutants Tom20 C13S and Tom20 C21S were separately transfected into cells. a Bax translocated to mitochondria upon CCCP/FeSO 4 stimulation as shown by confocal microscopy. b Knockdown of Tom20 blocked the translocation of Bax to mitochondria upon CCCP/FeSO 4 treatment. The mitochondrial fraction in cells was prepared. c Knockdown of Bax blocked the CCCP/FeSO 4 -induced mitochondrial aggregation. d-g After knocking down Bax, the CCCP/FeSO 4 -induced cytochrome c release detected in the cytosol fraction was diminished ( d ), the cleavage of caspase-3 and -9 was attenuated ( e ), the cell viability was rescued ( f ), the cell morphologies were reversed from pyroptosis to normal state, and LDH release and GSDME cleavage were also abolished ( g ). h Effects of the mutants Tom20 C13S and Tom20 C21S on the translocation of Bax to mitochondria in response to CCCP/FeSO 4 stimulation. Hsp60 was used to detect the mitochondria, and DAPI was used to display the nuclei by confocal microscopy. Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. *** P

    Journal: Cell Research

    Article Title: Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis

    doi: 10.1038/s41422-018-0090-y

    Figure Lengend Snippet: Tom20-induced translocation of Bax to mitochondria contributes to pyroptosis. Melanoma A375 cells were treated with CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 for 6 h to detect the translocation of Bax to mitochondria, cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. To detect the effects of the Tom20 point mutants Tom20 C13S and Tom20 C21S , Tom20 was knocked down in A375 cells, and Tom20 WT or its point mutants Tom20 C13S and Tom20 C21S were separately transfected into cells. a Bax translocated to mitochondria upon CCCP/FeSO 4 stimulation as shown by confocal microscopy. b Knockdown of Tom20 blocked the translocation of Bax to mitochondria upon CCCP/FeSO 4 treatment. The mitochondrial fraction in cells was prepared. c Knockdown of Bax blocked the CCCP/FeSO 4 -induced mitochondrial aggregation. d-g After knocking down Bax, the CCCP/FeSO 4 -induced cytochrome c release detected in the cytosol fraction was diminished ( d ), the cleavage of caspase-3 and -9 was attenuated ( e ), the cell viability was rescued ( f ), the cell morphologies were reversed from pyroptosis to normal state, and LDH release and GSDME cleavage were also abolished ( g ). h Effects of the mutants Tom20 C13S and Tom20 C21S on the translocation of Bax to mitochondria in response to CCCP/FeSO 4 stimulation. Hsp60 was used to detect the mitochondria, and DAPI was used to display the nuclei by confocal microscopy. Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. *** P

    Article Snippet: Caspase-3 (Cat# 707-C3-010/CF) was from R & D Systems.

    Techniques: Translocation Assay, Transfection, Confocal Microscopy

    A novel pathway of Tom20-Bax-caspase-GSDME upon iron stimulation. In melanoma cells, iron-elevated ROS causes the oxidation and oligomerization of Tom20. Oxidized Tom20 induces Bax translocation to mitochondria, which facilitates cytochrome c release to cytosol. Once released, cytochrome c activates caspase-9, which then activates caspase-3. This caspase-3 activation further cleaves GSDME, and eventually triggers cell swelling and LDH release

    Journal: Cell Research

    Article Title: Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis

    doi: 10.1038/s41422-018-0090-y

    Figure Lengend Snippet: A novel pathway of Tom20-Bax-caspase-GSDME upon iron stimulation. In melanoma cells, iron-elevated ROS causes the oxidation and oligomerization of Tom20. Oxidized Tom20 induces Bax translocation to mitochondria, which facilitates cytochrome c release to cytosol. Once released, cytochrome c activates caspase-9, which then activates caspase-3. This caspase-3 activation further cleaves GSDME, and eventually triggers cell swelling and LDH release

    Article Snippet: Caspase-3 (Cat# 707-C3-010/CF) was from R & D Systems.

    Techniques: Translocation Assay, Activation Assay

    Iron acts as a sensitizer for different drugs and induces pyroptosis in melanoma cells. Melanoma A375 cells were treated with SSZ (sulfasalazine, 125 μM) with or without FeSO 4 (100 μM) for 6 h to detect the ROS level or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release), unless specifically defined. a FeSO 4 acts as a sensitizer for ROS generation, GSDME cleavage, LDH release, and pyroptosis in the presence of SSZ. b , c Extensive treatment with SSZ/FeSO 4 at the indicated times induced Tom20 oxidation and accumulation ( b ) and GSDME cleavage ( c ). d – g Separate knockdown of Tom20, Bax, caspase-3 or GSDME blocked SSZ/FeSO 4 -induced pyroptosis, GSDME cleavage, and LDH release as indicated. Tubulin was used to determine the amount of loading proteins. All data are presented as the mean ± SEM of three independent experiments. ** P

    Journal: Cell Research

    Article Title: Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis

    doi: 10.1038/s41422-018-0090-y

    Figure Lengend Snippet: Iron acts as a sensitizer for different drugs and induces pyroptosis in melanoma cells. Melanoma A375 cells were treated with SSZ (sulfasalazine, 125 μM) with or without FeSO 4 (100 μM) for 6 h to detect the ROS level or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release), unless specifically defined. a FeSO 4 acts as a sensitizer for ROS generation, GSDME cleavage, LDH release, and pyroptosis in the presence of SSZ. b , c Extensive treatment with SSZ/FeSO 4 at the indicated times induced Tom20 oxidation and accumulation ( b ) and GSDME cleavage ( c ). d – g Separate knockdown of Tom20, Bax, caspase-3 or GSDME blocked SSZ/FeSO 4 -induced pyroptosis, GSDME cleavage, and LDH release as indicated. Tubulin was used to determine the amount of loading proteins. All data are presented as the mean ± SEM of three independent experiments. ** P

    Article Snippet: Caspase-3 (Cat# 707-C3-010/CF) was from R & D Systems.

    Techniques:

    Mitochondrial pathway with activation of caspase-3 is involved in pyroptosis induced by iron. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) or GSH (1 mM) for 2 h, followed by CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release), unless specifically defined. a Mitochondrial depletion blocked CCCP/FeSO 4 -induced pyroptosis, GSDME cleavage and LDH release. Mito: mitochondria. b CCCP/FeSO 4 induced mitochondrial accumulation, but NAC and GSH attenuated this accumulation. c CCCP/FeSO 4 induced cytochrome c release from mitochondria to cytosol as detected in the cytosol fraction (left) or by confocal microscopy (right). Cyto C: cytochrome c. d , e NAC or GSH abolished CCCP/FeSO 4 -induced cytochrome c release ( d ) and cleavage of caspase-3 and -9 ( e ). CASP: caspase. f Knockdown of FTL or FTH1 enhanced the CCCP/FeSO 4 -induced cleavage of caspase-3 and -9. g , h Knockdown of either caspase-3 or -9 abolished the CCCP/FeSO 4 -induced GSDME cleavage ( g ), pyroptosis and LDH release ( h ). Tubulin was used to determine the amount of loading proteins. All data are presented as the mean ± SEM of three independent experiments. ** P

    Journal: Cell Research

    Article Title: Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis

    doi: 10.1038/s41422-018-0090-y

    Figure Lengend Snippet: Mitochondrial pathway with activation of caspase-3 is involved in pyroptosis induced by iron. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) or GSH (1 mM) for 2 h, followed by CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release), unless specifically defined. a Mitochondrial depletion blocked CCCP/FeSO 4 -induced pyroptosis, GSDME cleavage and LDH release. Mito: mitochondria. b CCCP/FeSO 4 induced mitochondrial accumulation, but NAC and GSH attenuated this accumulation. c CCCP/FeSO 4 induced cytochrome c release from mitochondria to cytosol as detected in the cytosol fraction (left) or by confocal microscopy (right). Cyto C: cytochrome c. d , e NAC or GSH abolished CCCP/FeSO 4 -induced cytochrome c release ( d ) and cleavage of caspase-3 and -9 ( e ). CASP: caspase. f Knockdown of FTL or FTH1 enhanced the CCCP/FeSO 4 -induced cleavage of caspase-3 and -9. g , h Knockdown of either caspase-3 or -9 abolished the CCCP/FeSO 4 -induced GSDME cleavage ( g ), pyroptosis and LDH release ( h ). Tubulin was used to determine the amount of loading proteins. All data are presented as the mean ± SEM of three independent experiments. ** P

    Article Snippet: Caspase-3 (Cat# 707-C3-010/CF) was from R & D Systems.

    Techniques: Activation Assay, Confocal Microscopy

    ROS induces Tom20 oxidation in response to iron stimulation. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) for 2 h, followed by CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. To detect the effects of the Tom20 point mutants Tom20 C13S , Tom20 C21S and Tom20 C100S , Tom20 was knocked down in A375 cells, and Tom20 WT or its point mutants Tom20 C13S , Tom20 C21S and Tom20 C100S were separately transfected into cells. a FeSO 4 induced Tom20 oxidation, which was attenuated by NAC even in the presence of CCCP. Western blotting was performed under reducing and non-reducing conditions to determine the Tom20 oxidation status. b Mutation of either Cys13 or Cys21 in Tom20 abolished the CCCP/FeSO 4 -induced Tom20 oxidation. c CCCP/FeSO 4 could not induce Tom20 C13S and Tom20 C21S accumulation. d Mutation of either Cys13 or Cys21 in Tom20 blocked mitochondrial aggregation as shown by confocal microscopy. Hsp60 represents the mitochondria, and DAPI was used to display the nuclei. e-h Mutation of either Cys13 or Cys21 in Tom20 abrogated the CCCP/FeSO 4 -induced cytochrome c release as detected in the cytosol fraction ( e ), cleavage of caspase-3 and -9 ( f ), LDH release and cell death ( g ), and reversed the cell morphology from pyroptosis to normal state ( h ). Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. *** P

    Journal: Cell Research

    Article Title: Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis

    doi: 10.1038/s41422-018-0090-y

    Figure Lengend Snippet: ROS induces Tom20 oxidation in response to iron stimulation. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) for 2 h, followed by CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. To detect the effects of the Tom20 point mutants Tom20 C13S , Tom20 C21S and Tom20 C100S , Tom20 was knocked down in A375 cells, and Tom20 WT or its point mutants Tom20 C13S , Tom20 C21S and Tom20 C100S were separately transfected into cells. a FeSO 4 induced Tom20 oxidation, which was attenuated by NAC even in the presence of CCCP. Western blotting was performed under reducing and non-reducing conditions to determine the Tom20 oxidation status. b Mutation of either Cys13 or Cys21 in Tom20 abolished the CCCP/FeSO 4 -induced Tom20 oxidation. c CCCP/FeSO 4 could not induce Tom20 C13S and Tom20 C21S accumulation. d Mutation of either Cys13 or Cys21 in Tom20 blocked mitochondrial aggregation as shown by confocal microscopy. Hsp60 represents the mitochondria, and DAPI was used to display the nuclei. e-h Mutation of either Cys13 or Cys21 in Tom20 abrogated the CCCP/FeSO 4 -induced cytochrome c release as detected in the cytosol fraction ( e ), cleavage of caspase-3 and -9 ( f ), LDH release and cell death ( g ), and reversed the cell morphology from pyroptosis to normal state ( h ). Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. *** P

    Article Snippet: Caspase-3 (Cat# 707-C3-010/CF) was from R & D Systems.

    Techniques: Transfection, Western Blot, Mutagenesis, Confocal Microscopy

    Iron-induced Tom20 accumulation promotes pyroptosis. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) or GSH (1 mM) for 2 h, followed by CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. a Top, addition of FeSO 4 to CCCP induced Tom20 and Tom40 accumulation. Bottom, the effect of CCCP/FeSO 4 on the expression levels of Tom20 and Tom40. Cells were treated with CCCP or CCCP/FeSO 4 for the indicated times. b Knockdown of Tom20 rescued the cell viability in response to CCCP/FeSO 4 stimulation. c – e Knockdown of Tom20 reversed the CCCP/FeSO 4 -induced cell morphology from pyroptosis to normal state, reduced LDH release and blocked GSDME cleavage ( c ), abolished cytochrome c release (detected in the cytosol fraction) and cleavage of caspase-3 and -9 ( d ), and blocked mitochondria aggregation ( e ). Hsp60 was used as a mitochondrial indicator, and DAPI was used to display the nuclei. f NAC and GSH attenuated the CCCP/FeSO 4 -induced Tom20 accumulation. Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. *** P

    Journal: Cell Research

    Article Title: Tom20 senses iron-activated ROS signaling to promote melanoma cell pyroptosis

    doi: 10.1038/s41422-018-0090-y

    Figure Lengend Snippet: Iron-induced Tom20 accumulation promotes pyroptosis. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) or GSH (1 mM) for 2 h, followed by CCCP (20 μM), FeSO 4 (100 μM), or CCCP/FeSO 4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. a Top, addition of FeSO 4 to CCCP induced Tom20 and Tom40 accumulation. Bottom, the effect of CCCP/FeSO 4 on the expression levels of Tom20 and Tom40. Cells were treated with CCCP or CCCP/FeSO 4 for the indicated times. b Knockdown of Tom20 rescued the cell viability in response to CCCP/FeSO 4 stimulation. c – e Knockdown of Tom20 reversed the CCCP/FeSO 4 -induced cell morphology from pyroptosis to normal state, reduced LDH release and blocked GSDME cleavage ( c ), abolished cytochrome c release (detected in the cytosol fraction) and cleavage of caspase-3 and -9 ( d ), and blocked mitochondria aggregation ( e ). Hsp60 was used as a mitochondrial indicator, and DAPI was used to display the nuclei. f NAC and GSH attenuated the CCCP/FeSO 4 -induced Tom20 accumulation. Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. *** P

    Article Snippet: Caspase-3 (Cat# 707-C3-010/CF) was from R & D Systems.

    Techniques: Expressing

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

    Journal: Influenza and Other Respiratory Viruses

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

    doi: 10.1111/irv.12276

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

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

    Techniques: Activity Assay

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

    Journal: Influenza and Other Respiratory Viruses

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

    doi: 10.1111/irv.12276

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

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

    Techniques: Activity Assay

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.23.21.7829-7837.2003

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

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

    Techniques: Activation Assay, MTT Assay, Activity Assay

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.23.21.7829-7837.2003

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

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

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

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.23.21.7829-7837.2003

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

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

    Techniques: Activity Assay, Recombinant

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.23.21.7829-7837.2003

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

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

    Techniques: Incubation, Activity Assay, Inhibition

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

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.23.21.7829-7837.2003

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

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

    Techniques: Activation Assay, Incubation, Activity Assay

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

    Journal: Nanotheranostics

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

    doi: 10.7150/ntno.26389

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

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

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

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

    Journal: Nanotheranostics

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

    doi: 10.7150/ntno.26389

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

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

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

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

    Journal: ACS nano

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

    doi: 10.1021/nn504494c

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

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

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

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

    Journal: ACS nano

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

    doi: 10.1021/nn504494c

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

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

    Techniques: Histopathology, Staining

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

    Journal: ACS nano

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

    doi: 10.1021/nn504494c

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

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

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

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

    Journal: ACS nano

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

    doi: 10.1021/nn504494c

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

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

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