anti cleaved caspase 3 casp3  (Cell Signaling Technology Inc)

 
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    Name:
    Caspase 3 D3R6Y Rabbit mAb IHC Formulated
    Description:
    Caspase 3 CPP 32 Apoptain Yama SCA 1 is a critical executioner of apoptosis as it is either partially or totally responsible for the proteolytic cleavage of many key proteins such as the nuclear enzyme poly ADP ribose polymerase PARP 1 Activation of caspase 3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments Cleavage of caspase 3 requires the aspartic acid residue at the P1 position 2
    Catalog Number:
    14214
    Price:
    None
    Applications:
    Immunohistochemistry
    Category:
    Primary Antibodies
    Source:
    Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the p20 subunit of human caspase-3 protein.
    Reactivity:
    Human
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    Structured Review

    Cell Signaling Technology Inc anti cleaved caspase 3 casp3
    Oligodendrocyte precursor cell susceptibility to apoptotic induction following inflammatory stress. Analysis of <t>Casp3</t> expression in O4+ cells following an inflammatory stress. The fold change of apoptotic (Casp3+) and viable (Casp3-) O4+ cells was similar in the WT and Hp70.1 KO CNS cell cultures after 24 h of stimulation with LPS plus IFN-γ. The error bars correspond to the SEM.
    Caspase 3 CPP 32 Apoptain Yama SCA 1 is a critical executioner of apoptosis as it is either partially or totally responsible for the proteolytic cleavage of many key proteins such as the nuclear enzyme poly ADP ribose polymerase PARP 1 Activation of caspase 3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments Cleavage of caspase 3 requires the aspartic acid residue at the P1 position 2
    https://www.bioz.com/result/anti cleaved caspase 3 casp3/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti cleaved caspase 3 casp3 - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Hsp70 Regulates Immune Response in Experimental Autoimmune Encephalomyelitis"

    Article Title: Hsp70 Regulates Immune Response in Experimental Autoimmune Encephalomyelitis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105737

    Oligodendrocyte precursor cell susceptibility to apoptotic induction following inflammatory stress. Analysis of Casp3 expression in O4+ cells following an inflammatory stress. The fold change of apoptotic (Casp3+) and viable (Casp3-) O4+ cells was similar in the WT and Hp70.1 KO CNS cell cultures after 24 h of stimulation with LPS plus IFN-γ. The error bars correspond to the SEM.
    Figure Legend Snippet: Oligodendrocyte precursor cell susceptibility to apoptotic induction following inflammatory stress. Analysis of Casp3 expression in O4+ cells following an inflammatory stress. The fold change of apoptotic (Casp3+) and viable (Casp3-) O4+ cells was similar in the WT and Hp70.1 KO CNS cell cultures after 24 h of stimulation with LPS plus IFN-γ. The error bars correspond to the SEM.

    Techniques Used: Expressing

    Apoptotic induction in CNS cell cultures. Apoptotic immunocytochemical detection in oligodendrocyte precursor cells (OPCs, O4+ cells) and mature oligodendrocytes (MBP+ cells) in mixed CNS cell cultures of WT and Hsp70.1 KO mice under non-stimulated (left column) and LPS plus IFN-γ-stimulated (right column) culture conditions. A. Detection of apoptotic OPCs cells (O4+Casp3+ cells). The rows indicate the O4+Casp3+ cells. B. Detection of apoptotic mature oligodendrocytes (MBP+Casp3+ cells).
    Figure Legend Snippet: Apoptotic induction in CNS cell cultures. Apoptotic immunocytochemical detection in oligodendrocyte precursor cells (OPCs, O4+ cells) and mature oligodendrocytes (MBP+ cells) in mixed CNS cell cultures of WT and Hsp70.1 KO mice under non-stimulated (left column) and LPS plus IFN-γ-stimulated (right column) culture conditions. A. Detection of apoptotic OPCs cells (O4+Casp3+ cells). The rows indicate the O4+Casp3+ cells. B. Detection of apoptotic mature oligodendrocytes (MBP+Casp3+ cells).

    Techniques Used: Mouse Assay

    2) Product Images from "Unilateral Blood Flow Decrease Induces Bilateral and Symmetric Responses in the Immature Brain"

    Article Title: Unilateral Blood Flow Decrease Induces Bilateral and Symmetric Responses in the Immature Brain

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2009.090257

    Neonatal ischemia triggers activation of caspase-3 (casp3) in both hemispheres. Cleaved casp3 immunolabeling (red) and TUNEL assay (DNA fragmentation, green) in sections of brain at the level of the dorsal hippocampus (bregma −3.3 mm) from contralateral (CL-M1, A–C ) and ipsilateral (IL-M1, D–F ) hemispheres at 48 hours postischemia (model M1). Note that active casp3 was cytosolic in the CL cortex (enlarged panel in C ), whereas it was nuclear and cytosolic and associated with TUNEL staining in the IL cortex (enlarged panel in F ). Scale bar represents 50 and 20 μm in enlarged panels. G: Spatial distribution of cleaved casp3- (red) and TUNEL-positive (green) cells and lesion area (gray) at 48 hours after ischemic injury in P7 rats in both IL and CL hemispheres. H: Representative Western blots probed with anti-active casp3 (17 kDa; Cell Signaling Technology) for protein samples isolated from the cytosolic (S2) and nuclear (P1) fractions of IL and CL cortex from ischemic rats sacrificed 48 hours after reperfusion and from sham (Sh) brain. The cytosolic marker β-actin was used as protein loading control. Note that p17 was present in the cytosolic fractions in both IL and CL tissues. In contrast, p17 was only present in the nuclear fraction for IL tissues; Fr, fraction. I: Quantification of casp3-positive cells at 4, 12, 48, and 72 hours postischemia in both IL and CL cortex.
    Figure Legend Snippet: Neonatal ischemia triggers activation of caspase-3 (casp3) in both hemispheres. Cleaved casp3 immunolabeling (red) and TUNEL assay (DNA fragmentation, green) in sections of brain at the level of the dorsal hippocampus (bregma −3.3 mm) from contralateral (CL-M1, A–C ) and ipsilateral (IL-M1, D–F ) hemispheres at 48 hours postischemia (model M1). Note that active casp3 was cytosolic in the CL cortex (enlarged panel in C ), whereas it was nuclear and cytosolic and associated with TUNEL staining in the IL cortex (enlarged panel in F ). Scale bar represents 50 and 20 μm in enlarged panels. G: Spatial distribution of cleaved casp3- (red) and TUNEL-positive (green) cells and lesion area (gray) at 48 hours after ischemic injury in P7 rats in both IL and CL hemispheres. H: Representative Western blots probed with anti-active casp3 (17 kDa; Cell Signaling Technology) for protein samples isolated from the cytosolic (S2) and nuclear (P1) fractions of IL and CL cortex from ischemic rats sacrificed 48 hours after reperfusion and from sham (Sh) brain. The cytosolic marker β-actin was used as protein loading control. Note that p17 was present in the cytosolic fractions in both IL and CL tissues. In contrast, p17 was only present in the nuclear fraction for IL tissues; Fr, fraction. I: Quantification of casp3-positive cells at 4, 12, 48, and 72 hours postischemia in both IL and CL cortex.

    Techniques Used: Activation Assay, Immunolabeling, TUNEL Assay, Staining, Western Blot, Isolation, Marker

    Unilateral transient CCA occlusion mainly triggers casp3 cleavage in neurons and undifferentiated cells in the P7 rat brain. Contralateral cortex from uni-CCAo (model M2) and MCAo + tCCAo (model M1) animals contain most cleaved casp3 colocalized with neurons (NeuN) ( A , B ), nestin- ( C , D ), and vimentin ( E , F ) positive cells Arrows indicate location magnified in insets . Co-localization of cleaved casp3 was observed in GABA-immunostained neurons in cortical layer III (ly III in G , and enlarged panels in H , indicated by the asterisk in G ). Cleaved casp3 labeling is shown in red, whereas NeuN, nestin, vimentin and GABA markers are shown in green. Scale bar represents 50 and 20 μm (enlarged panels).
    Figure Legend Snippet: Unilateral transient CCA occlusion mainly triggers casp3 cleavage in neurons and undifferentiated cells in the P7 rat brain. Contralateral cortex from uni-CCAo (model M2) and MCAo + tCCAo (model M1) animals contain most cleaved casp3 colocalized with neurons (NeuN) ( A , B ), nestin- ( C , D ), and vimentin ( E , F ) positive cells Arrows indicate location magnified in insets . Co-localization of cleaved casp3 was observed in GABA-immunostained neurons in cortical layer III (ly III in G , and enlarged panels in H , indicated by the asterisk in G ). Cleaved casp3 labeling is shown in red, whereas NeuN, nestin, vimentin and GABA markers are shown in green. Scale bar represents 50 and 20 μm (enlarged panels).

    Techniques Used: Labeling

    Casp3 cleavage and DNA fragmentation in several models of carotid occlusion at 48 hours after injury. Spatial distribution ( A , E , and I ) of cleaved casp3 (red), TUNEL-positive (green) cells, and lesion area (gray) after uni-CCAo (model M2), bi-tCCAo (model M3) and bi-pCCAo (model M4), respectively. Note that images in mirror were detected in all three ischemic conditions. Double fluorescent staining in the CL cortex for cleaved casp3 ( B , F , and J ) and TUNEL ( C , top , G , and K ) or Fluorojade B (FluJB) labeling ( C , bottom ). After uni-CCAo, only cytosolic cleaved casp3 (enlarged image in D ) without TUNEL or FluJB staining was observed. In contrast, several cells displayed cytosolic cleaved casp3 co-localized with TUNEL staining ( F–H , note chromatin clumps typical of apoptotic cells, enlarged panel in H ) after model M3. A large number of cells exhibited both cytosolic and nuclear cleaved casp3 and DNA fragmentation after model M4 ( J–L ). Some sections were counterstained with 4′,6′-diamidino-2-phenylindole (overlay in D and L ). Scale bar represents 50 and 20 μm (enlarged panels).
    Figure Legend Snippet: Casp3 cleavage and DNA fragmentation in several models of carotid occlusion at 48 hours after injury. Spatial distribution ( A , E , and I ) of cleaved casp3 (red), TUNEL-positive (green) cells, and lesion area (gray) after uni-CCAo (model M2), bi-tCCAo (model M3) and bi-pCCAo (model M4), respectively. Note that images in mirror were detected in all three ischemic conditions. Double fluorescent staining in the CL cortex for cleaved casp3 ( B , F , and J ) and TUNEL ( C , top , G , and K ) or Fluorojade B (FluJB) labeling ( C , bottom ). After uni-CCAo, only cytosolic cleaved casp3 (enlarged image in D ) without TUNEL or FluJB staining was observed. In contrast, several cells displayed cytosolic cleaved casp3 co-localized with TUNEL staining ( F–H , note chromatin clumps typical of apoptotic cells, enlarged panel in H ) after model M3. A large number of cells exhibited both cytosolic and nuclear cleaved casp3 and DNA fragmentation after model M4 ( J–L ). Some sections were counterstained with 4′,6′-diamidino-2-phenylindole (overlay in D and L ). Scale bar represents 50 and 20 μm (enlarged panels).

    Techniques Used: TUNEL Assay, Staining, Labeling

    Unilateral transient CCA occlusion can induce casp3 cleavage but not its downstream subtracts. A: Representative Western blots showing cleaved casp3 fragment (17 kDa) both in the IL and CL 48 hours after uni-tCCAo + MCAo (model M1) and after uni-CCAo (model M2) as compared with sham-operated neocortical cytosolic extracts ( n = 8 for each condition). Highly cleaved casp3 was present in the IL after model M1, but it also is increased in the CL cortex and after model M2 compared with sham (sh) cytosolic extracts ( P
    Figure Legend Snippet: Unilateral transient CCA occlusion can induce casp3 cleavage but not its downstream subtracts. A: Representative Western blots showing cleaved casp3 fragment (17 kDa) both in the IL and CL 48 hours after uni-tCCAo + MCAo (model M1) and after uni-CCAo (model M2) as compared with sham-operated neocortical cytosolic extracts ( n = 8 for each condition). Highly cleaved casp3 was present in the IL after model M1, but it also is increased in the CL cortex and after model M2 compared with sham (sh) cytosolic extracts ( P

    Techniques Used: Western Blot

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    Article Snippet: .. The membranes were blocked for 1 h with 50 g/l skim milk in TBST washing buffer (20 mM Tris, 150 mM NaCl, and 0.1% Tween 20) and then incubated overnight with primary antibodies at 4°C, followed by incubation with designated secondary antibodies for 1 h. In this study, the employed primary antibodies were illustrated as following: anti-β-actin, anti-LC3, anti-PARP, and anti-cleaved PARP (Gentex, Irvine, CA, USA); anti-phospho-Akt (Ser473), anti-cleaved caspase-3, and anti-cleaved caspase-9 (Cell Signaling Technology, Danvers, MA, USA). ..

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    Article Snippet: .. The membranes were incubated overnight at 4°C with a 1:1,000 solution of primary rabbit antibodies: Anti-E-cadherin (Abcam, Cambridge, UK; cat. no. ab15148), anti-β-catenin (Abcam; cat. no. ab16051), anti-vimentin (Abcam; cat. no. ab92547), anti-cluster of differentiation (CD)63 (Abcam; cat. no. ab134045), anti-CD81 (Abcam; cat. no. ab109201), anti-cleaved poly(ADP-ribose) polymerase (PARP; cat. no. 5625; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-cleaved caspase-3 (cat. no. 9664; Cell Signaling Technology, Inc.), anti-apoptosis regulator Bcl-2 (Bcl-2; cat. no. 4223, Cell Signaling Technology, Inc.), anti-apoptosis regulator BAX (Bax; cat. no. 5023; Cell Signaling Technology, Inc.), and anti-β-actin (Abcam; cat. no. ab8227). .. The horseradish peroxidase-conjugated anti-rabbit antibody (cat. no. 7074; 1:5,000; Cell Signaling Technology, Inc.) was used as secondary antibody for immunostaining for 1 h at room temperature.

    Article Title: miR-338-3p functions as a tumor suppressor in gastric cancer by targeting PTP1B
    Article Snippet: .. The membranes were blocked with 5% non-fat dry milk and then incubated with primary antibody: anti-PTP1B and anti-GAPDH (sc-14021 and sc-47724, respectively; Santa Cruz, Dallas, TX, USA); anti-β-actin (ab8227; Abcam, Cambridge, UK); anti-Caspase-3, anti-cleaved Caspase-3, anti-AKT, anti-p-AKT, anti-ERK1/2, anti-p-ERK1/2 (9662, 9664, 9272, 4060, 4695, 4370, respectively; Cell Signaling Technology, Inc., MA, USA). .. Bound antibodies with anti-rabbit secondary antibodies (Santa Cruz) were detected and visualized by Pierce chemiluminescent substrate (Thermo Fisher).

    Article Title: Caspase-2 deficiency accelerates chemically induced liver cancer in mice
    Article Snippet: .. Non-specific antibody binding was blocked using and 5% (vol/vol) FBS/PBS solution with Tween-20 for 30 min. Tissue sections were incubated with anti-cleaved caspase-3 (9664; Cell Signaling Technology, Beverly, MA, USA) diluted 1:200, anti-glypican3 (ab66596; Abcam, Cambridge, MA, USA) diluted 1:200, anti-PCNA (clone PC10; Cell Signaling Technology) diluted 1:250 and anti-phospho histone γ H2AX (Ser139) (Cell Signaling) primary antibody diluted 1:100 in blocking solution overnight at 4 °C. .. Tissue sections were sequentially incubated with anti-mouse or anti-rabbit biotinylated secondary antibody diluted 1:250 in blocking solution (GE Healthcare, Waukesha, WI, USA) and Avidin/Biotin Complex reagent (VectaStain ABC kit, Vector Labs, Burlingame, CA, USA) at room temperature.

    Binding Assay:

    Article Title: Caspase-2 deficiency accelerates chemically induced liver cancer in mice
    Article Snippet: .. Non-specific antibody binding was blocked using and 5% (vol/vol) FBS/PBS solution with Tween-20 for 30 min. Tissue sections were incubated with anti-cleaved caspase-3 (9664; Cell Signaling Technology, Beverly, MA, USA) diluted 1:200, anti-glypican3 (ab66596; Abcam, Cambridge, MA, USA) diluted 1:200, anti-PCNA (clone PC10; Cell Signaling Technology) diluted 1:250 and anti-phospho histone γ H2AX (Ser139) (Cell Signaling) primary antibody diluted 1:100 in blocking solution overnight at 4 °C. .. Tissue sections were sequentially incubated with anti-mouse or anti-rabbit biotinylated secondary antibody diluted 1:250 in blocking solution (GE Healthcare, Waukesha, WI, USA) and Avidin/Biotin Complex reagent (VectaStain ABC kit, Vector Labs, Burlingame, CA, USA) at room temperature.

    Immunohistochemistry:

    Article Title: Survival signal REG3 α prevents crypt apoptosis to control acute gastrointestinal graft-versus-host disease
    Article Snippet: .. Immunohistochemistry was performed with polyclonal rabbit anti-REG3α (Abcam, ab134309) at a 1:200 dilution for human samples and rabbit anti-REG3γ from the Lora V. Hooper laboratory ( ) at a 1:8,000 dilution or rabbit anti–cleaved caspase-3 (clone 5A1E, Cell Signaling) at a 1:400 dilution for murine samples using a DAKO AutoStainer Link; slides were subsequently coated with a goat anti-rabbit IgG HRP conjugate (DAKO) at a 1:200 dilution and finally a diaminobenzidine (DAB) dilution to generate brown-colored signals. ..

    Blocking Assay:

    Article Title: Caspase-2 deficiency accelerates chemically induced liver cancer in mice
    Article Snippet: .. Non-specific antibody binding was blocked using and 5% (vol/vol) FBS/PBS solution with Tween-20 for 30 min. Tissue sections were incubated with anti-cleaved caspase-3 (9664; Cell Signaling Technology, Beverly, MA, USA) diluted 1:200, anti-glypican3 (ab66596; Abcam, Cambridge, MA, USA) diluted 1:200, anti-PCNA (clone PC10; Cell Signaling Technology) diluted 1:250 and anti-phospho histone γ H2AX (Ser139) (Cell Signaling) primary antibody diluted 1:100 in blocking solution overnight at 4 °C. .. Tissue sections were sequentially incubated with anti-mouse or anti-rabbit biotinylated secondary antibody diluted 1:250 in blocking solution (GE Healthcare, Waukesha, WI, USA) and Avidin/Biotin Complex reagent (VectaStain ABC kit, Vector Labs, Burlingame, CA, USA) at room temperature.

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  • 88
    Cell Signaling Technology Inc rabbit anti casp3
    Premature apoptosis occured in C9ORF72 spinal MNs over ageing (a) Apoptosis in MAP2-positive neurons (in red) was revealed as cytosolic rim staining around Hoechst-positive nuclei (in blue) for cleaved caspase 3 <t>(Casp3,</t> in green) by confocal IF microscopy at D80 endpoints (fig. 3). Dotted boxed areas in image galleries are shown magnified on the right. Note the striking accumulation of Casp3 in parental C9 and C9-KO that was phenocopied by WT-KO. Conversely, apoptosis hardly occurred in C9-GC and parental Ctrl1. Scale bars = 10µm. (b) Quantification of (a), percentage of apoptotic cells in MAP2-positive population. Asterisks: highly significant increase in any pairwise comparison with unlabeled conditions, one-way ANOVA with Bonferroni post test, *P≤0.05, **P≤0.01, ***P≤0.001, N=60 images from 3 independent experiments, error bars=SD. All unlabeled conditions (i.e. with no asterisk) were not significantly different amongst themselves in any pairwise comparison. Remaining comparisons were: WT-KO vs C9: ***, WT-KO vs C9-KO: ***, C9 vs C9-KO: ***.
    Rabbit Anti Casp3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc anti caspase 3
    TRAP1 C501S mutant exerts protective role towards staurosporine-induced cell death A ) Western blot analyses of HeLa cell lysates were performed to check the expression levels of WT and C501S mutant forms of GFP-fused TRAP1 (TRAP1-GFP) which can be discriminated by the endogenous variant (TRAP1) by the use of an anti-TRAP1 antibody. Western blot shown is representative of at least n = 3 independent experiments giving similar results.  B ) The number of dead cells was evaluated by Trypan blue exclusion test in control cells transfected with an empty vector (Mock), as well as in cell expressing TRAP1 WT  (WT) and TRAP1 C501S  (C501S) treated for 4 h with 200 nM staurosporine (STS), in the presence or absence of the NO donor DETA-NONOate (250 μM, maintained for 22-24 h before STS treatment). Data are shown as fold change of dead cells with respect to untreated cells, and represent the mean ± SEM of n =5 independent experiments done in duplicate. * p  = 0.05; ** p  = 0.01.  C ) Western blot analyses of HeLa cells treated as in (B) were performed to evaluate cleaved PARP1 and pro-caspase 3 (Casp3) protein levels. Vinculin and α-actin were used as loading controls. Densitometric analyses (denso) of PARP1/Vinculin or Casp3/actin are shown below each lane. Western blots shown are representative of at least n = 3 independent experiments giving similar results.
    Anti Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 769 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc anti cleaved caspase 3
    <t>Caspase</t> 3 staining in kidney sections and isolated podocytes. A) A Immunohistochemical staining with anti-caspase 3 antibody. Scale bar: 50μm (upper row); 20μm (lower row). B) Confocal micrographs from primary podocytes derived from described genotypes showing cytoplasm staining (GAPDH in green), apoptotic cells (Cleaved CASPASE-3 in red) and nuclei (DAPI in blue). Maximum intensity projection of Z-stacks is shown. Scale bars: 50 μm. OnOn the right panel, qquantification of caspase 3-positive cells. Data are presented as mean ± SD. Symbols * and § represent the significance levels vs. WT and Mpv17 -/- , respectively, calculated by one‐way ANOVA with Tukey’s post hoc multiple comparison test − : **** p
    Anti Cleaved Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc cleaved caspase 3
    SARS-CoV-2 internalizes and replicates within hiPSC-CMs in vitro , eliciting cytopathic effect and contractility alterations. A) Human iPSC-CMs exhibit standard sarcomeric markers including cardiac troponin T (cTnT) and α-actinin with DAPI as nuclear counterstain. B) Immunofluorescence for cTnT and SARS-CoV-2 “spike” protein demonstrates that hiPSC-CMs can be infected by SARS-CoV-2. SARS-CoV-2 spike protein is not present in mock infected cultures. C) HiPSC-CMs after SARS-CoV-2 infection, but not mock infection, exhibit signs of cellular apoptosis, indicated by morphological changes seen in bright field (BF) and cleaved <t>caspase-3</t> production. A second SARS-CoV-2 antibody marks a viral-specific double-stranded intermediate RNA (dsRNA). D) Magnified inset from panel B shows a merged immunofluorescence image for SARS-CoV-2 spike protein and DAPI. Arrows indicate perinuclear accumulation of viral particles, and suggests active viral protein translation and genome replication at perinuclear ribosomes and membranous compartments. E) Magnified inset from panel C shows a merged immunofluorescence image for SARS-CoV-2 dsRNA and DAPI. Arrows indicate perinuclear viral replication sites. F) Quantification of immunofluorescence indicates percentage of total DAPI-positive cells that are positive for spike protein, viral dsRNA, cleaved caspase-3 (CC3), and dsRNA+CC3 in hiPSC-CMs infected with SARS-CoV-2, compared to mock infection. N=5-7 images quantified for each stain for mock and infected conditions. G) Quantification of beats per minute in wells containing hiPSC-CMs with mock infection versus wells containing hiPSC-CMs infected with SARS-CoV-2. N=6 videos recorded for each condition. See Supplemental Movie 1 for representative video clips. * indicates p
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    Image Search Results


    Premature apoptosis occured in C9ORF72 spinal MNs over ageing (a) Apoptosis in MAP2-positive neurons (in red) was revealed as cytosolic rim staining around Hoechst-positive nuclei (in blue) for cleaved caspase 3 (Casp3, in green) by confocal IF microscopy at D80 endpoints (fig. 3). Dotted boxed areas in image galleries are shown magnified on the right. Note the striking accumulation of Casp3 in parental C9 and C9-KO that was phenocopied by WT-KO. Conversely, apoptosis hardly occurred in C9-GC and parental Ctrl1. Scale bars = 10µm. (b) Quantification of (a), percentage of apoptotic cells in MAP2-positive population. Asterisks: highly significant increase in any pairwise comparison with unlabeled conditions, one-way ANOVA with Bonferroni post test, *P≤0.05, **P≤0.01, ***P≤0.001, N=60 images from 3 independent experiments, error bars=SD. All unlabeled conditions (i.e. with no asterisk) were not significantly different amongst themselves in any pairwise comparison. Remaining comparisons were: WT-KO vs C9: ***, WT-KO vs C9-KO: ***, C9 vs C9-KO: ***.

    Journal: bioRxiv

    Article Title: High content live profiling reveals concomitant gain and loss of function pathomechanisms in C9ORF72 amyotrophic lateral sclerosis

    doi: 10.1101/2020.04.15.040394

    Figure Lengend Snippet: Premature apoptosis occured in C9ORF72 spinal MNs over ageing (a) Apoptosis in MAP2-positive neurons (in red) was revealed as cytosolic rim staining around Hoechst-positive nuclei (in blue) for cleaved caspase 3 (Casp3, in green) by confocal IF microscopy at D80 endpoints (fig. 3). Dotted boxed areas in image galleries are shown magnified on the right. Note the striking accumulation of Casp3 in parental C9 and C9-KO that was phenocopied by WT-KO. Conversely, apoptosis hardly occurred in C9-GC and parental Ctrl1. Scale bars = 10µm. (b) Quantification of (a), percentage of apoptotic cells in MAP2-positive population. Asterisks: highly significant increase in any pairwise comparison with unlabeled conditions, one-way ANOVA with Bonferroni post test, *P≤0.05, **P≤0.01, ***P≤0.001, N=60 images from 3 independent experiments, error bars=SD. All unlabeled conditions (i.e. with no asterisk) were not significantly different amongst themselves in any pairwise comparison. Remaining comparisons were: WT-KO vs C9: ***, WT-KO vs C9-KO: ***, C9 vs C9-KO: ***.

    Article Snippet: The following primary antibodies were used: mouse anti-yH2A.X (1:500, Millipore #05- 636), rabbit anti-53BP1 (1:1000, Novusbio NB100-304), chicken anti-MAP2 (1:1000, Abcam ab5392), rabbit anti-Casp3 (1:1000, Cell Signaling #9661), rat anti-GP (1:500, clone 18H8) and mouse anti GA (1:500, clone IAI2) were both generously provided from Dieter Edbauer ( ).

    Techniques: Staining, Microscopy

    TRAP1 C501S mutant exerts protective role towards staurosporine-induced cell death A ) Western blot analyses of HeLa cell lysates were performed to check the expression levels of WT and C501S mutant forms of GFP-fused TRAP1 (TRAP1-GFP) which can be discriminated by the endogenous variant (TRAP1) by the use of an anti-TRAP1 antibody. Western blot shown is representative of at least n = 3 independent experiments giving similar results.  B ) The number of dead cells was evaluated by Trypan blue exclusion test in control cells transfected with an empty vector (Mock), as well as in cell expressing TRAP1 WT  (WT) and TRAP1 C501S  (C501S) treated for 4 h with 200 nM staurosporine (STS), in the presence or absence of the NO donor DETA-NONOate (250 μM, maintained for 22-24 h before STS treatment). Data are shown as fold change of dead cells with respect to untreated cells, and represent the mean ± SEM of n =5 independent experiments done in duplicate. * p  = 0.05; ** p  = 0.01.  C ) Western blot analyses of HeLa cells treated as in (B) were performed to evaluate cleaved PARP1 and pro-caspase 3 (Casp3) protein levels. Vinculin and α-actin were used as loading controls. Densitometric analyses (denso) of PARP1/Vinculin or Casp3/actin are shown below each lane. Western blots shown are representative of at least n = 3 independent experiments giving similar results.

    Journal: bioRxiv

    Article Title: S-nitrosylation affects TRAP1 structure and ATPase activity and modulates cell response to apoptotic stimuli

    doi: 10.1101/859629

    Figure Lengend Snippet: TRAP1 C501S mutant exerts protective role towards staurosporine-induced cell death A ) Western blot analyses of HeLa cell lysates were performed to check the expression levels of WT and C501S mutant forms of GFP-fused TRAP1 (TRAP1-GFP) which can be discriminated by the endogenous variant (TRAP1) by the use of an anti-TRAP1 antibody. Western blot shown is representative of at least n = 3 independent experiments giving similar results. B ) The number of dead cells was evaluated by Trypan blue exclusion test in control cells transfected with an empty vector (Mock), as well as in cell expressing TRAP1 WT (WT) and TRAP1 C501S (C501S) treated for 4 h with 200 nM staurosporine (STS), in the presence or absence of the NO donor DETA-NONOate (250 μM, maintained for 22-24 h before STS treatment). Data are shown as fold change of dead cells with respect to untreated cells, and represent the mean ± SEM of n =5 independent experiments done in duplicate. * p = 0.05; ** p = 0.01. C ) Western blot analyses of HeLa cells treated as in (B) were performed to evaluate cleaved PARP1 and pro-caspase 3 (Casp3) protein levels. Vinculin and α-actin were used as loading controls. Densitometric analyses (denso) of PARP1/Vinculin or Casp3/actin are shown below each lane. Western blots shown are representative of at least n = 3 independent experiments giving similar results.

    Article Snippet: We used the following primary antibodies: anti-HSP75 (TRAP1) and anti-Actin from Santa Cruz Biotechnology; anti-Vinculin from Sigma-Aldrich; anti-PARP1 from Enzo Life Sciences; anti-Caspase 3 from Cell Signaling.

    Techniques: Mutagenesis, Western Blot, Expressing, Variant Assay, Transfection, Plasmid Preparation

    Caspase 3 staining in kidney sections and isolated podocytes. A) A Immunohistochemical staining with anti-caspase 3 antibody. Scale bar: 50μm (upper row); 20μm (lower row). B) Confocal micrographs from primary podocytes derived from described genotypes showing cytoplasm staining (GAPDH in green), apoptotic cells (Cleaved CASPASE-3 in red) and nuclei (DAPI in blue). Maximum intensity projection of Z-stacks is shown. Scale bars: 50 μm. OnOn the right panel, qquantification of caspase 3-positive cells. Data are presented as mean ± SD. Symbols * and § represent the significance levels vs. WT and Mpv17 -/- , respectively, calculated by one‐way ANOVA with Tukey’s post hoc multiple comparison test − : **** p

    Journal: bioRxiv

    Article Title: Opa1 overexpression protects from early onset Mpv17-/--related mouse kidney disease

    doi: 10.1101/2020.03.18.996561

    Figure Lengend Snippet: Caspase 3 staining in kidney sections and isolated podocytes. A) A Immunohistochemical staining with anti-caspase 3 antibody. Scale bar: 50μm (upper row); 20μm (lower row). B) Confocal micrographs from primary podocytes derived from described genotypes showing cytoplasm staining (GAPDH in green), apoptotic cells (Cleaved CASPASE-3 in red) and nuclei (DAPI in blue). Maximum intensity projection of Z-stacks is shown. Scale bars: 50 μm. OnOn the right panel, qquantification of caspase 3-positive cells. Data are presented as mean ± SD. Symbols * and § represent the significance levels vs. WT and Mpv17 -/- , respectively, calculated by one‐way ANOVA with Tukey’s post hoc multiple comparison test − : **** p

    Article Snippet: Antibodies Mouse monoclonal anti-GAPDH (1:1,000; #ab8245; Abcam), rabbit monoclonal anti-TOM20 Alexa Fluor 647 (1:500; #ab209606; Abcam), Alexa Fluor 488 Goat anti-mouse (1:300; #A11004; Invitrogen), Alexa Fluor 647 Goat anti-rabbit (1:300; #A27040; Invitrogen), rabbit polyclonal anti-NPHS2 (1:300; #ab50339; Abcam), mouse monoclonal anti-CYTOKERATIN (1:300; #ab86734; Abcam), anti-cleaved caspase 3 (1:200; Cell Signalling, #9661).

    Techniques: Staining, Isolation, Immunohistochemistry, Derivative Assay

    SARS-CoV-2 internalizes and replicates within hiPSC-CMs in vitro , eliciting cytopathic effect and contractility alterations. A) Human iPSC-CMs exhibit standard sarcomeric markers including cardiac troponin T (cTnT) and α-actinin with DAPI as nuclear counterstain. B) Immunofluorescence for cTnT and SARS-CoV-2 “spike” protein demonstrates that hiPSC-CMs can be infected by SARS-CoV-2. SARS-CoV-2 spike protein is not present in mock infected cultures. C) HiPSC-CMs after SARS-CoV-2 infection, but not mock infection, exhibit signs of cellular apoptosis, indicated by morphological changes seen in bright field (BF) and cleaved caspase-3 production. A second SARS-CoV-2 antibody marks a viral-specific double-stranded intermediate RNA (dsRNA). D) Magnified inset from panel B shows a merged immunofluorescence image for SARS-CoV-2 spike protein and DAPI. Arrows indicate perinuclear accumulation of viral particles, and suggests active viral protein translation and genome replication at perinuclear ribosomes and membranous compartments. E) Magnified inset from panel C shows a merged immunofluorescence image for SARS-CoV-2 dsRNA and DAPI. Arrows indicate perinuclear viral replication sites. F) Quantification of immunofluorescence indicates percentage of total DAPI-positive cells that are positive for spike protein, viral dsRNA, cleaved caspase-3 (CC3), and dsRNA+CC3 in hiPSC-CMs infected with SARS-CoV-2, compared to mock infection. N=5-7 images quantified for each stain for mock and infected conditions. G) Quantification of beats per minute in wells containing hiPSC-CMs with mock infection versus wells containing hiPSC-CMs infected with SARS-CoV-2. N=6 videos recorded for each condition. See Supplemental Movie 1 for representative video clips. * indicates p

    Journal: bioRxiv

    Article Title: Human iPSC-Derived Cardiomyocytes are Susceptible to SARS-CoV-2 Infection

    doi: 10.1101/2020.04.21.051912

    Figure Lengend Snippet: SARS-CoV-2 internalizes and replicates within hiPSC-CMs in vitro , eliciting cytopathic effect and contractility alterations. A) Human iPSC-CMs exhibit standard sarcomeric markers including cardiac troponin T (cTnT) and α-actinin with DAPI as nuclear counterstain. B) Immunofluorescence for cTnT and SARS-CoV-2 “spike” protein demonstrates that hiPSC-CMs can be infected by SARS-CoV-2. SARS-CoV-2 spike protein is not present in mock infected cultures. C) HiPSC-CMs after SARS-CoV-2 infection, but not mock infection, exhibit signs of cellular apoptosis, indicated by morphological changes seen in bright field (BF) and cleaved caspase-3 production. A second SARS-CoV-2 antibody marks a viral-specific double-stranded intermediate RNA (dsRNA). D) Magnified inset from panel B shows a merged immunofluorescence image for SARS-CoV-2 spike protein and DAPI. Arrows indicate perinuclear accumulation of viral particles, and suggests active viral protein translation and genome replication at perinuclear ribosomes and membranous compartments. E) Magnified inset from panel C shows a merged immunofluorescence image for SARS-CoV-2 dsRNA and DAPI. Arrows indicate perinuclear viral replication sites. F) Quantification of immunofluorescence indicates percentage of total DAPI-positive cells that are positive for spike protein, viral dsRNA, cleaved caspase-3 (CC3), and dsRNA+CC3 in hiPSC-CMs infected with SARS-CoV-2, compared to mock infection. N=5-7 images quantified for each stain for mock and infected conditions. G) Quantification of beats per minute in wells containing hiPSC-CMs with mock infection versus wells containing hiPSC-CMs infected with SARS-CoV-2. N=6 videos recorded for each condition. See Supplemental Movie 1 for representative video clips. * indicates p

    Article Snippet: The following antibodies and dilutions were used: α-actinin (1:100, Sigma-Aldrich Cat# A7811, RRID: AB_476766); cardiac troponin T (cTnT, 1:100, Abcam Cat# ab45932, RRID: AB_956386); SARS-CoV-2 spike (S) protein (1:100, BEI Resources NR-616 Monoclonal Anti-SARS-CoV S Protein (Similar to 240C) SARS coronavirus); SARS-CoV-2 double stranded RNA (1:100, J2 clone; Absolute Antibody Inc.); cleaved caspase-3 (1:200, Cell Signaling Technology Cat# 9661, RRID: AB_2341188).

    Techniques: In Vitro, Immunofluorescence, Infection, Staining