caspase 8 Search Results


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  • 97
    Millipore caspase 8
    <t>Caspase</t> 8 is up-regulated in Smad7-expressing MCF7 cells. A , total RNA was isolated from control or FLAG-Smad7-expressing MCF7 cells, and semi-quantitative RT-PCR was performed using caspase 8 ( Casp8 )- or Smad7-specific primers. GAPDH was used as internal
    Caspase 8, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1311 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc caspase 8
    The combination of panobinostat plus marizomib induces greater and earlier caspase-3 activity and is <t>caspase-8</t> dependent in ML-1 cells A B) ML-1 cells were pre-treated with inhibitors of caspase-8 (A, IETD-fmk) or caspase-9 (B, LEHD-fmk), followed by treatment with combinations of panobinostat with marizomib or bortezomib. DNA fragmentation was assessed by flow cytometry after propidium iodide staining. C) ML-1 cells were treated for 12 hours with 1 μM panobinostat, 10 nM bortezomib, and 50 nM marizomib. Lysates were probed for cleaved caspase-3. D) ML-1 cells were treated for indicated times with either panobinostat (1 μM) plus marizomib (50 nM) or panobinostat (1 μM) plus bortezomib (10 nM) combinations. Caspase-3/7 activity was measured using the fluorogenic substrate DEVD-amc. E) After 16 hours of treatment with each of the combinations (1 μM panobinostat plus 50 nM marizomib or 1 μM panobinostat plus 10 nM bortezomib), cells were stained with HEt for analysis of superoxide levels. F) ML-1 cells were pretreated for 30 minutes with 24 mM NAC, followed by 24 hours of treatment with diluent, 10 nM bortezomib, 50 nM marizomib, 1 μM panobinostat, or combinations of these agents. DNA fragmentation was assessed by PI staining (*p
    Caspase 8, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 5126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc cleaved caspase 8
    Constitutive CXCL12 expression induces anoikis through an intrinsic apoptotic mechanism. (A) CXCL12 and GFP HT29 cells were grown for 4-days as a suspension on poly-HEMA [10 mg/ml]-treated plates (Sus.) and whole cell lysates collected and compared to cells grown as an adherent monolayer (Adh.). Active cleaved <t>caspase-8,</t> -3, -7, and -9 indicated the predominant activation of an intrinsic apoptotic pathway in CXCL12 cells relative to control. Gliotoxin [2 µg/ml] confirmed intact apoptotic machinery in both cell lines. (B,C) Luminescence-based caspase activity assays were used to examine levels of both initiator caspases-8 and -9 and executioner caspases-3/7 after 4-days on poly-HEMA. Clonal HT29 (B) and HCT116 (C) cell lines expressing variable levels of CXCL12 (Hi, Med, Low) indicated a dose dependent effect of caspase activity which was inhibited with addition of zVAD. Values are mean ± SD of 3 independent experiments completed in triplicate. (D) GFP and CXCL12 HT29 cells were cultured on poly-HEMA-treated plates and lysates collected over time. Immunoblot analysis revealed a decrease in Mcl-1 and Bcl-2 levels and an increase in Bim in CXCL12-expressing cells. Arrowhead indicates a lower molecular weight protein consistent with an Mcl-1 degradation product. (E) HT29 cells expressing variable levels of CXCL12 exhibit increased Bim expression after a 4-day culture on poly-HEMA. (F) Immunoblot analysis of adherent HT29 cells demonstrate the temporal loss of FAK activation precedes decreased Mcl-1 levels in CXCL12-expressing cells cultured as an adherent monolayer on tissue culture plastic. Data are representative of three independent experiments. * indicates statistically significant differences in caspase activity between GFP and CXCL12-expressing cells ( P ≤0.05).
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    92
    Santa Cruz Biotechnology caspase 8
    Schematic of protein kinase Cδ (PKCδ) activation by proteolytic cleavage downstream of tumor necrosis factor (TNF) signaling in dopaminergic neurons. (1) Sustained TNF signaling leads to <t>caspase-8</t> activation, presumably by autoproteolytic cleavage at the receptor complex (2) . Active caspase-8 leads to downstream activation of caspase-3, which can be blocked by the caspase-8 inhibitor IETD-fmk (3) . Caspase-3 activates PKCδ by proteolytic cleavage (4) at the hinge region (shown in red), releasing the catalytic fragment and causing constitutive activation of the kinase (5) . The constitutively active PKCδ catalytic fragment mediates proapoptotic signaling by phosphorylation of its downstream substrates, resulting in DNA fragmentation and dopaminergic cell death (6) . Blocking the PKCδ signaling pathway using siRNA, targeted gene knockout or a caspase-3 cleavage-resistant mutant can protect against dopaminergic cell death induced by TNF toxicity.
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    93
    Cell Signaling Technology Inc anti caspase 8
    Styrene oxide induced the onset of apoptosis. The subconfluence C2C12 myoblasts were treated with styrene or styrene oxide at the indicated concentrations in a growth medium for 48 h. After treatment, the nucleus was stained with Hoechst 43332 (a) and nuclear size was measured (b). The apoptosis markers in styrene oxide (c-d) or styrene (e-f) treatment were detected by Western blot with anti-caspase-9, <t>anti-caspase-8,</t> and anti-caspase-3 antibodies. ∗ p
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    99
    Abcam caspase 8
    In vivo anticancer effects of TIPE-2 (6.0 mg/kg) treatment on hepatocellular carcinoma-bearing mice. (A) Antitumor effects of TIPE-2 treatment on HepG2-bearing nude mice. (B) TIPE-2 treatment promoted apoptosis of cells within tumor tissues. (C) TIPE-2 treatment increased the expression levels of caspase-3 and <t>caspase-8</t> in tumor tissues. Effects of TIPE-2 treatment on the expression levels of (D) PI3K and AKT, and (E) GRP78 and CHOP in tumor tissues. Magnification, ×40. (F) Survival rate of HepG2-bearing mice following treatment with TIPE-2 or PBS. **P
    Caspase 8, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 750 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    R&D Systems caspase 8
    Images of cells transfected with M mRNA or treated with inhibitors of host gene expression in the presence of a <t>caspase-8</t> inhibitor. HeLa cells were untreated (A) or transfected with yeast RNA (B) as negative controls. HeLa cells were treated with TRAIL as a positive control (C and D), transfected with M mRNA (E and F), treated with actinomycin D (G and H), or treated with DRB (I and J). Cells shown in panels D, F, H, and J were pretreated with 100 μM synthetic caspase-8 inhibitor, Z-IETD-FMK. Phase-contrast images were captured with a ×20 objective. Representative images were chosen from three separate experiments.
    Caspase 8, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 480 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson caspase 8
    Confocal microscopy study of the binding of Bid and <t>caspase-8</t> to giant unilamellar vesicles containing cardiolipin. Trios of images (top, middle and bottom) for the same sample: two images obtained with two different detector channels of the microscope, together with an overlay image. DOPC-only (100∶0) vesicles are presented in panels a to c and DOPC/CL (90∶10) vesicles in panels d to f . Top: in a and d , protein binding to GUVs shown in green (this binding only becomes apparent when the green label accumulates at the membrane); middle: the GUV membrane was labelled with 0.05% of the hydrophobic dye DiO, as shown in ( b, c ) and in red, as shown in ( e, f ); bottom: overlay of green and red images ( c, f ). Time is indicated in minutes. The arrows indicate the decrease in GUV fluorescence following the formation of a complex between procaspase-8 and Bid Alexa488 , resulting in a non-fluorescent tBid.
    Caspase 8, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 587 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti cleaved caspase 8
    Increased RIP1/Caspase 8 containing complexes in SMCs exposing to primed RAWs and aneurysmal tissues. ( A ) SMCs were exposed to MCP-1 primed RAWs or naïve RAWs for 3 days. Left: RIP1/Caspase 8 containing complex formation was examined by in situ proximity ligation assay (PLA). Middle: co-immunostaining for SMCs (SM-αA, green) and RIP1/Caspase 8 containing complex (red spots). Nuclei (DAPI, blue). Right: magnified view of the boxed areas in the middle panel. Scale bar, 100 μm. ( B ) Increased RIP1/Caspase 8 containing complex in elastase-treated aortas. Arteries were harvested 3 days after aneurysm induction by elastase infusion. Heat-inactivated elastase served as a control. Nuclei (DAPI, blue), SMCs (SM-αA, green), RIP1/Caspase 8 containing complex (red spots). Higher magnified views of highlighted regions were shown on the right. L indicates lumen. Scale bar, 50 μm.
    Anti Cleaved Caspase 8, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 451 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology anti caspase 8
    The expression and activation of apoptosis-associated molecules in GBM cells treated with BP. Whole cell lysates (20 µg/lane) were analyzed with western blotting using specific antibodies to: (a) p53, phospho-p53 and phospho-RB; (b) p16, p21, p27, cdk2, cdk4, cdk6, cyclin D1 and cyclin E; (c) Bax, AIF and caspase 9; (d) Fas, Fas-L, <t>caspase</t> 8 and caspase 3. Lower panel: Relation to control in (a) to (d) is relative to untreated control cells. Positive control of RG2 cells is DBTRG-05 MG cells with BP treatment for 3 h. UD, protein undetectable in this western blot system: ND, not detected.
    Anti Caspase 8, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem caspase 8
    Effect of EG on the expression of apoptosis-associated proteins in HL-60 cells. HL-60 cells (5 × 10 5 cells) were treated with 50 μM or 75 μM EG for 6 h, 12 h, or 24 h. Cell lysates were resolved by SDS-PAGE and subjected to western blotting. ( A ) Bcl-2, Bax, and tBid. Con: Control, Cyto: cytosol, Mito: mitochondria; ( B ) <t>Caspase-8,</t> caspase-9, caspase-3, apoptosis-inducing factor (AIF), and Endo G; ( C ) Cytochrome c . These results presented are representative of data obtained from three independent experiments carried out in triplicate. These results presented are representative of data obtained from three independent experiments carried out in triplicate.
    Caspase 8, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 450 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher caspase 8
    Representative photomicrographs at 10× magnification with 20× magnification inset panel and scale bar representing 100 μm. a Caspase-3 high expression; b caspase-3 low expression; c <t>caspase-8</t> high expression; d caspase-8 low expression
    Caspase 8, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 810 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen caspase 8
    Effects of <t>caspase-8</t> downregulation in skin maturation. A. Immunohistochemistry analysis performed in skin from wildtype and caspase-8 knock out mice. Caspase-8 DEDs are stained in green, keratin 5 in red and nuclei in blue. Brackets denote expanded epidermis in the caspase-8 knockout mice. B. Microtubule staining (red channel) in skin from wild type and caspase-8 knockout mice. Insets show magnified views of the microtubule patterning in wild type and caspase-8 knockout mice. C. Left, Confocal images of HaCat undifferentiated and differentiated cells stained for caspase-8 DEDs (green channel) and microtubules (red channel). Yellow arrows indicate centrosomes. Right, Immunoblot analysis of caspase-8 expression in undifferentiated or differentiated keratinocytes. Lysates were probed with antibody recognizing the DEDs of caspase-8.
    Caspase 8, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc mouse anti caspase 8
    Src interacts with PP2A/C and <t>caspase-8,</t> and TRAIL treatment promotes their interaction. A , Src interacts with PP2A/C and caspase-8 ( Casp8 ). 293T cells were co-transfected with pcDNA3-Src (Src), caspase-8-HA, and PP2A/C-HA. After 48 h, cells were left
    Mouse Anti Caspase 8, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    BioVision caspase 8
    Role of caspases in cell death induction by MSA/paclitaxel. A. Western blot of PARP cleavage by MSA/paclitaxel. B. Western blot of cleaved <t>caspase-8</t> and -9 in the presence of MSA or paclitaxel, or the combination. C. Increase of cleaved caspase-8 and caspase-9 by MSA, paclitaxel, or combination. D. Effect of caspase-8 inhibitor or caspase-9 inhibitor on cell death induction by MSA/paclitaxel. *Statistically different (P
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    99
    Millipore caspase 3 colorimetric activity assay kit
    Activation of apoptosis in xenograft tumors and cancer cells with suppressed levels of NAF-1. (A) Left, immunohistochemistry (IHC) analysis using an antibody against activated <t>caspase-3</t> showing a higher number of active-caspase-3-positive cells in NAF-1(−) tumors. Active-caspase-3-positive cells are marked by white arrowheads. Right, quantification of staining of active caspase-3. (B) Left, IHC analysis using an antibody against γH2AX showing a higher number of γH2AX-positive cells in NAF-1(−) tumors. Right, quantification of γH2AX staining. For A,B, cells were counted in ten high-power fields (×40) for each section obtained from five mice in each group; *** P
    Caspase 3 Colorimetric Activity Assay Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 364 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Novus Biologicals caspase 8
    <t>Caspase</t> 8 gene ablation provides neuroprotection in vitro in primary neuronal cultures and brain organotypic cultures. ( A–B ) 3-day-old PNC derived from embryos (E16.5) of CRE3 ( A ) and N casp8 −/− homozygous mice ( B ) were treated with recombinant murine TNFα and cycloheximide (CHX), then fixed and stained with Hoechst dye and immunostained with an antibody to NeuN followed by application of Alexa Fluor 594 conjugated anti-mouse antibody. ( C1–J4 ). Untreated (UNT C1–F4 ), STS-treated, ( G1–H4 ) and recombinant murine TNFα and CHX-treated ( I1–J4 ) 14-day-old neuronal cultures derived from CRE3 and N casp8 −/− mice were preincubated with pSIVA reagent (green) for 15 min prior to Z-fix fixation, then immunostained for MAP2 (red) and stained with DAPI (blue). To assess neuronal phenotype, the transmission light, single ( C1, D1, E4, G1, H1, H3, I1, I2, I4, J1 ), double ( C2, D2, E1, E2, F1, F3, G4, H2, H4, I3, J2 ), and triple ( E3, F2, F4, G2, G3, J3, J4 ) channel images are provided. Colocalization of neuronal marker MAP2 (red) with pSIVA signal (green) resulted in yellow color denoting degenerating neurons whose number was counted and plotted as ratio to the total number of neurons ( Figure 2B ). Single and multichannel panels confirm specificity of the double labeling. PSIVA was visualized from the early (membranous staining) to later stages (staining in the cell body and nucleus, the latter in cells with loss of plasma cell integrity) of neuronal degeneration.
    Caspase 8, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 89/100, based on 134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore caspase 8 inhibitor
    Y465 phosphomimetic modification of caspase-8A is necessary but not sufficient for Src activation. A) HEK293 cells were co-transfected with Y465E GFP-caspase-8A mutant and Y527F Src for 24 hours. Whole cell lysates were subjected to Western blot analysis with an anti-phosphotyrosine antibody (non-reactive to glutamic acid residue in phosphomimetic mutant) and an anti-GFP antibody. Y465E of GFP-caspase-8A was tyrosine phosphorylated. B) HEK293 cells were co-transfected with Y465E GFP-caspase-8A mutant and Y527F Src for 24 hours. GFP-caspase-8A IP was sent for LC-MS/MS analysis. C) This is a schematic figure showing the location of tyrosine residues that we mutated. D, E) We transfected HEK293 cells with Y527F Src and various GFP-caspase-8A mutants for 24 hours. Whole cell lysates were subjected to Western blot analysis with pY416Src antibody and Src antibody. N = 4, p = 0.001 (Y465E vs Y397E/Y465F); p = 0.139 (Y465E vs Y397F/Y465E). F, G) We transfected HEK293 cells with Y527F Src and pEGFP empty plasmid or pEGFP fusion plasmid with WT or various mutants of <t>GFP-caspase-8</t> for 24 hours. We then immunoprecipitated transfected avian Src and resolve the immunoprecipitate on SDS-PAGE for Western blot analysis. N = 3, p = 0.04.
    Caspase 8 Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson anti caspase 8
    Model depicting the etoposide-induced apoptotic signaling pathway in SK-N-AS cells. Etoposide induces the mitochondrial cytochrome c release, leading to the caspase-9-dependent activation of caspase-3. The activation of caspase-3 induces the cleavage of PKCδ, and active PKCδ processes caspase-3 by a positive-feedback mechanism. The activation of caspase-3 leads to the processing of <t>caspase-8,</t> and the expression of caspase-8 and caspase-2 is required for the activation of each other downstream of caspase-3. The etoposide-induced activation of caspase-8 leads to the processing of caspase-6 and apoptosis. Rottlerin inhibits etoposide-induced apoptotic signaling by preventing the PKCδ-mediated activation of caspase-3 and by causing the degradation of caspase-2, which inhibits caspase-8 activation.
    Anti Caspase 8, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime caspase 8
    Inactivation of caspase-dependent apoptosis in ischemic cardiomyocytes treated with an AKT2 inhibitor. ( A ) Initiator <t>caspase-8</t> activity in extracts of cardiomyocytes with con, an AKT2 inhibitor, and ischemic cardiomyocytes treated with or without AKT2 inhibitor and/or zVAD; ( B ) Quantitative real time PCR of cFlip transcript in cardiomyocytes with the same treatment as in A. zVAD: pan-caspase inhibitor z-VAD-fmk, 100 μM. The bars represent the mean ± SEM of three independent experiments. * p
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    Enzo Biochem anti caspase 8
    <t>Caspase-8</t> and -9 are processed in F. novicida -infected Casp1 KO macrophages and their inhibition reverts caspase-1-independent cell death. Caspase-8 ( a ) and -9 ( b ) cleavage were investigated by immunoblot analysis in WT, Casp1 KO and ASC KO BMM infected
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    Image Search Results


    Caspase 8 is up-regulated in Smad7-expressing MCF7 cells. A , total RNA was isolated from control or FLAG-Smad7-expressing MCF7 cells, and semi-quantitative RT-PCR was performed using caspase 8 ( Casp8 )- or Smad7-specific primers. GAPDH was used as internal

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M112.400408

    Figure Lengend Snippet: Caspase 8 is up-regulated in Smad7-expressing MCF7 cells. A , total RNA was isolated from control or FLAG-Smad7-expressing MCF7 cells, and semi-quantitative RT-PCR was performed using caspase 8 ( Casp8 )- or Smad7-specific primers. GAPDH was used as internal

    Article Snippet: For knockdown of caspase 8, we used pLKO.1-puro lentiviral short hairpin RNA (shRNA) system according to the manufacturer's instructions (Sigma).

    Techniques: Expressing, Isolation, Quantitative RT-PCR

    Smad7 and IRF1 cooperate to induce caspase 8 expression. A , to test the involvement of IRF1-binding site, ISRE reporter assay was performed in MCF7 cells. After transfection with ISRE reporter construct with Smad7, cells were lysed to measure the luciferase

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M112.400408

    Figure Lengend Snippet: Smad7 and IRF1 cooperate to induce caspase 8 expression. A , to test the involvement of IRF1-binding site, ISRE reporter assay was performed in MCF7 cells. After transfection with ISRE reporter construct with Smad7, cells were lysed to measure the luciferase

    Article Snippet: For knockdown of caspase 8, we used pLKO.1-puro lentiviral short hairpin RNA (shRNA) system according to the manufacturer's instructions (Sigma).

    Techniques: Expressing, Binding Assay, Reporter Assay, Transfection, Construct, Luciferase

    Involvement of Smad7 in IFN-γ-induced caspase 8 expression. A , total RNA was isolated in scrambled or Smad7 siRNA-transfected cells after treatment with 50 ng/ml IFN-γ for 48 h. Expression of caspase 8 ( Casp8 ) or Smad7 was detected by

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M112.400408

    Figure Lengend Snippet: Involvement of Smad7 in IFN-γ-induced caspase 8 expression. A , total RNA was isolated in scrambled or Smad7 siRNA-transfected cells after treatment with 50 ng/ml IFN-γ for 48 h. Expression of caspase 8 ( Casp8 ) or Smad7 was detected by

    Article Snippet: For knockdown of caspase 8, we used pLKO.1-puro lentiviral short hairpin RNA (shRNA) system according to the manufacturer's instructions (Sigma).

    Techniques: Expressing, Isolation, Transfection

    Caspase 8 is required for TRAIL-mediated apoptosis in MCF7 cells. A , MCF7 cells were treated with 100 ng/ml TRAIL for 24 h, and protein lysates were prepared as described under “Experimental Procedures.” A total of 30 μg of protein

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M112.400408

    Figure Lengend Snippet: Caspase 8 is required for TRAIL-mediated apoptosis in MCF7 cells. A , MCF7 cells were treated with 100 ng/ml TRAIL for 24 h, and protein lysates were prepared as described under “Experimental Procedures.” A total of 30 μg of protein

    Article Snippet: For knockdown of caspase 8, we used pLKO.1-puro lentiviral short hairpin RNA (shRNA) system according to the manufacturer's instructions (Sigma).

    Techniques:

    Smad7 regulates transcription of caspase 8. A , to understand the mechanism of induced expression of caspase 8 in MCF7-Smad7 cells, we performed the luciferase assays using caspase 8 promoter-luciferase containing the promoter region (−1615/+16).

    Journal: The Journal of Biological Chemistry

    Article Title:

    doi: 10.1074/jbc.M112.400408

    Figure Lengend Snippet: Smad7 regulates transcription of caspase 8. A , to understand the mechanism of induced expression of caspase 8 in MCF7-Smad7 cells, we performed the luciferase assays using caspase 8 promoter-luciferase containing the promoter region (−1615/+16).

    Article Snippet: For knockdown of caspase 8, we used pLKO.1-puro lentiviral short hairpin RNA (shRNA) system according to the manufacturer's instructions (Sigma).

    Techniques: Expressing, Luciferase

    The combination of panobinostat plus marizomib induces greater and earlier caspase-3 activity and is caspase-8 dependent in ML-1 cells A B) ML-1 cells were pre-treated with inhibitors of caspase-8 (A, IETD-fmk) or caspase-9 (B, LEHD-fmk), followed by treatment with combinations of panobinostat with marizomib or bortezomib. DNA fragmentation was assessed by flow cytometry after propidium iodide staining. C) ML-1 cells were treated for 12 hours with 1 μM panobinostat, 10 nM bortezomib, and 50 nM marizomib. Lysates were probed for cleaved caspase-3. D) ML-1 cells were treated for indicated times with either panobinostat (1 μM) plus marizomib (50 nM) or panobinostat (1 μM) plus bortezomib (10 nM) combinations. Caspase-3/7 activity was measured using the fluorogenic substrate DEVD-amc. E) After 16 hours of treatment with each of the combinations (1 μM panobinostat plus 50 nM marizomib or 1 μM panobinostat plus 10 nM bortezomib), cells were stained with HEt for analysis of superoxide levels. F) ML-1 cells were pretreated for 30 minutes with 24 mM NAC, followed by 24 hours of treatment with diluent, 10 nM bortezomib, 50 nM marizomib, 1 μM panobinostat, or combinations of these agents. DNA fragmentation was assessed by PI staining (*p

    Journal: Leukemia research

    Article Title: EFFICACY OF PANOBINOSTAT AND MARIZOMIB IN ACUTE MYELOID LEUKEMIA AND BORTEZOMIB-RESISTANT MODELS

    doi: 10.1016/j.leukres.2014.12.014

    Figure Lengend Snippet: The combination of panobinostat plus marizomib induces greater and earlier caspase-3 activity and is caspase-8 dependent in ML-1 cells A B) ML-1 cells were pre-treated with inhibitors of caspase-8 (A, IETD-fmk) or caspase-9 (B, LEHD-fmk), followed by treatment with combinations of panobinostat with marizomib or bortezomib. DNA fragmentation was assessed by flow cytometry after propidium iodide staining. C) ML-1 cells were treated for 12 hours with 1 μM panobinostat, 10 nM bortezomib, and 50 nM marizomib. Lysates were probed for cleaved caspase-3. D) ML-1 cells were treated for indicated times with either panobinostat (1 μM) plus marizomib (50 nM) or panobinostat (1 μM) plus bortezomib (10 nM) combinations. Caspase-3/7 activity was measured using the fluorogenic substrate DEVD-amc. E) After 16 hours of treatment with each of the combinations (1 μM panobinostat plus 50 nM marizomib or 1 μM panobinostat plus 10 nM bortezomib), cells were stained with HEt for analysis of superoxide levels. F) ML-1 cells were pretreated for 30 minutes with 24 mM NAC, followed by 24 hours of treatment with diluent, 10 nM bortezomib, 50 nM marizomib, 1 μM panobinostat, or combinations of these agents. DNA fragmentation was assessed by PI staining (*p

    Article Snippet: To verify the role for caspase-8 activation as an early event in panobinostat-induced cell death, we measured cleavage of caspase-8 in RPMI-8226vr10 cells ( ).

    Techniques: Activity Assay, Flow Cytometry, Cytometry, Staining

    Constitutive CXCL12 expression induces anoikis through an intrinsic apoptotic mechanism. (A) CXCL12 and GFP HT29 cells were grown for 4-days as a suspension on poly-HEMA [10 mg/ml]-treated plates (Sus.) and whole cell lysates collected and compared to cells grown as an adherent monolayer (Adh.). Active cleaved caspase-8, -3, -7, and -9 indicated the predominant activation of an intrinsic apoptotic pathway in CXCL12 cells relative to control. Gliotoxin [2 µg/ml] confirmed intact apoptotic machinery in both cell lines. (B,C) Luminescence-based caspase activity assays were used to examine levels of both initiator caspases-8 and -9 and executioner caspases-3/7 after 4-days on poly-HEMA. Clonal HT29 (B) and HCT116 (C) cell lines expressing variable levels of CXCL12 (Hi, Med, Low) indicated a dose dependent effect of caspase activity which was inhibited with addition of zVAD. Values are mean ± SD of 3 independent experiments completed in triplicate. (D) GFP and CXCL12 HT29 cells were cultured on poly-HEMA-treated plates and lysates collected over time. Immunoblot analysis revealed a decrease in Mcl-1 and Bcl-2 levels and an increase in Bim in CXCL12-expressing cells. Arrowhead indicates a lower molecular weight protein consistent with an Mcl-1 degradation product. (E) HT29 cells expressing variable levels of CXCL12 exhibit increased Bim expression after a 4-day culture on poly-HEMA. (F) Immunoblot analysis of adherent HT29 cells demonstrate the temporal loss of FAK activation precedes decreased Mcl-1 levels in CXCL12-expressing cells cultured as an adherent monolayer on tissue culture plastic. Data are representative of three independent experiments. * indicates statistically significant differences in caspase activity between GFP and CXCL12-expressing cells ( P ≤0.05).

    Journal: PLoS ONE

    Article Title: CXCL12 Chemokine Expression and Secretion Regulates Colorectal Carcinoma Cell Anoikis through Bim-Mediated Intrinsic Apoptosis

    doi: 10.1371/journal.pone.0012895

    Figure Lengend Snippet: Constitutive CXCL12 expression induces anoikis through an intrinsic apoptotic mechanism. (A) CXCL12 and GFP HT29 cells were grown for 4-days as a suspension on poly-HEMA [10 mg/ml]-treated plates (Sus.) and whole cell lysates collected and compared to cells grown as an adherent monolayer (Adh.). Active cleaved caspase-8, -3, -7, and -9 indicated the predominant activation of an intrinsic apoptotic pathway in CXCL12 cells relative to control. Gliotoxin [2 µg/ml] confirmed intact apoptotic machinery in both cell lines. (B,C) Luminescence-based caspase activity assays were used to examine levels of both initiator caspases-8 and -9 and executioner caspases-3/7 after 4-days on poly-HEMA. Clonal HT29 (B) and HCT116 (C) cell lines expressing variable levels of CXCL12 (Hi, Med, Low) indicated a dose dependent effect of caspase activity which was inhibited with addition of zVAD. Values are mean ± SD of 3 independent experiments completed in triplicate. (D) GFP and CXCL12 HT29 cells were cultured on poly-HEMA-treated plates and lysates collected over time. Immunoblot analysis revealed a decrease in Mcl-1 and Bcl-2 levels and an increase in Bim in CXCL12-expressing cells. Arrowhead indicates a lower molecular weight protein consistent with an Mcl-1 degradation product. (E) HT29 cells expressing variable levels of CXCL12 exhibit increased Bim expression after a 4-day culture on poly-HEMA. (F) Immunoblot analysis of adherent HT29 cells demonstrate the temporal loss of FAK activation precedes decreased Mcl-1 levels in CXCL12-expressing cells cultured as an adherent monolayer on tissue culture plastic. Data are representative of three independent experiments. * indicates statistically significant differences in caspase activity between GFP and CXCL12-expressing cells ( P ≤0.05).

    Article Snippet: Antibodies specific for Bax, Bak, Mcl-1, Bim, phospho-Bad, total Bad, Puma, Bcl-2, Bcl-XL , cleaved poly-ADP ribose polymerase (PARP), cleaved caspase-3, cleaved caspase-9, cleaved caspase-8, and cleaved caspase-7 were from (Cell Signaling, Danvers, MA, USA).

    Techniques: Expressing, Activation Assay, Activity Assay, Cell Culture, Molecular Weight

    Schematic of protein kinase Cδ (PKCδ) activation by proteolytic cleavage downstream of tumor necrosis factor (TNF) signaling in dopaminergic neurons. (1) Sustained TNF signaling leads to caspase-8 activation, presumably by autoproteolytic cleavage at the receptor complex (2) . Active caspase-8 leads to downstream activation of caspase-3, which can be blocked by the caspase-8 inhibitor IETD-fmk (3) . Caspase-3 activates PKCδ by proteolytic cleavage (4) at the hinge region (shown in red), releasing the catalytic fragment and causing constitutive activation of the kinase (5) . The constitutively active PKCδ catalytic fragment mediates proapoptotic signaling by phosphorylation of its downstream substrates, resulting in DNA fragmentation and dopaminergic cell death (6) . Blocking the PKCδ signaling pathway using siRNA, targeted gene knockout or a caspase-3 cleavage-resistant mutant can protect against dopaminergic cell death induced by TNF toxicity.

    Journal: Journal of Neuroinflammation

    Article Title: Proteolytic activation of proapoptotic kinase protein kinase C? by tumor necrosis factor ? death receptor signaling in dopaminergic neurons during neuroinflammation

    doi: 10.1186/1742-2094-9-82

    Figure Lengend Snippet: Schematic of protein kinase Cδ (PKCδ) activation by proteolytic cleavage downstream of tumor necrosis factor (TNF) signaling in dopaminergic neurons. (1) Sustained TNF signaling leads to caspase-8 activation, presumably by autoproteolytic cleavage at the receptor complex (2) . Active caspase-8 leads to downstream activation of caspase-3, which can be blocked by the caspase-8 inhibitor IETD-fmk (3) . Caspase-3 activates PKCδ by proteolytic cleavage (4) at the hinge region (shown in red), releasing the catalytic fragment and causing constitutive activation of the kinase (5) . The constitutively active PKCδ catalytic fragment mediates proapoptotic signaling by phosphorylation of its downstream substrates, resulting in DNA fragmentation and dopaminergic cell death (6) . Blocking the PKCδ signaling pathway using siRNA, targeted gene knockout or a caspase-3 cleavage-resistant mutant can protect against dopaminergic cell death induced by TNF toxicity.

    Article Snippet: The results from our mechanistic studies using N27 rat dopaminergic cells indeed demonstrate for the first time that soluble TNF induces a time-dependent and dose-dependent proteolytic cleavage of PKCδ by a caspase-8 and caspase-3 signaling pathway, indicative of canonical TNF death receptor signaling via caspase-8.

    Techniques: Activation Assay, Blocking Assay, Gene Knockout, Mutagenesis

    Tumor necrosis factor (TNF) induced proteolytic cleavage of protein kinase Cδ (PKCδ) in dopaminergic N27 cells. (A) Time-dependent PKCδ proteolytic activation. N27 dopaminergic cells were treated with recombinant TNF (30 ng/ml) for 3 h and 6 h and lysates were probed by Western blotting with an antibody to the C-terminus that detects both the native protein (78 kDa) and the proteolytically cleaved catalytic fragment (38 kDa). TNF treatment caused a time-dependent increase in PKCδ cleavage, which peaked at 6 h. (B) Band intensities for cleaved PKCδ from three blots quantified using densitometric analysis and normalized to β-actin. (C) Dose-dependent proteolytic activation of PKCδ by TNF treatment. N27 cells were treated with increasing doses of TNF (0 to 60 ng/ml) for 6 h and lysates were probed for PKCδ by Western blotting. TNF induced a dose dependent increase in PKCδ cleavage, starting at 10 ng/ml. (D) Band intensities for cleaved PKCδ from three blots were quantified using densitometric analysis and normalized to β-actin. (E) Caspase-3 and caspase-8 dependent proteolytic cleavage of PKCδ. N27 cells were treated with 30 ng/ml of TNF alone for 6 h or in the presence of peptide inhibitors (25 μM) of caspase-8 (Ac-IETD-fmk) and caspase-3 (Ac-DEVD-fmk). Lysates were probed for PKCδ proteolytic cleavage by Western blotting. Proteolytic activation of PKCδ by TNF treatment was reduced by inhibition of caspase-3 and caspase-8. (F) Band intensities for cleaved PKCδ from three blots were quantified using densitometric analysis and normalized to β-actin. Data is expressed as a fold change over control. * ( P

    Journal: Journal of Neuroinflammation

    Article Title: Proteolytic activation of proapoptotic kinase protein kinase C? by tumor necrosis factor ? death receptor signaling in dopaminergic neurons during neuroinflammation

    doi: 10.1186/1742-2094-9-82

    Figure Lengend Snippet: Tumor necrosis factor (TNF) induced proteolytic cleavage of protein kinase Cδ (PKCδ) in dopaminergic N27 cells. (A) Time-dependent PKCδ proteolytic activation. N27 dopaminergic cells were treated with recombinant TNF (30 ng/ml) for 3 h and 6 h and lysates were probed by Western blotting with an antibody to the C-terminus that detects both the native protein (78 kDa) and the proteolytically cleaved catalytic fragment (38 kDa). TNF treatment caused a time-dependent increase in PKCδ cleavage, which peaked at 6 h. (B) Band intensities for cleaved PKCδ from three blots quantified using densitometric analysis and normalized to β-actin. (C) Dose-dependent proteolytic activation of PKCδ by TNF treatment. N27 cells were treated with increasing doses of TNF (0 to 60 ng/ml) for 6 h and lysates were probed for PKCδ by Western blotting. TNF induced a dose dependent increase in PKCδ cleavage, starting at 10 ng/ml. (D) Band intensities for cleaved PKCδ from three blots were quantified using densitometric analysis and normalized to β-actin. (E) Caspase-3 and caspase-8 dependent proteolytic cleavage of PKCδ. N27 cells were treated with 30 ng/ml of TNF alone for 6 h or in the presence of peptide inhibitors (25 μM) of caspase-8 (Ac-IETD-fmk) and caspase-3 (Ac-DEVD-fmk). Lysates were probed for PKCδ proteolytic cleavage by Western blotting. Proteolytic activation of PKCδ by TNF treatment was reduced by inhibition of caspase-3 and caspase-8. (F) Band intensities for cleaved PKCδ from three blots were quantified using densitometric analysis and normalized to β-actin. Data is expressed as a fold change over control. * ( P

    Article Snippet: The results from our mechanistic studies using N27 rat dopaminergic cells indeed demonstrate for the first time that soluble TNF induces a time-dependent and dose-dependent proteolytic cleavage of PKCδ by a caspase-8 and caspase-3 signaling pathway, indicative of canonical TNF death receptor signaling via caspase-8.

    Techniques: Activation Assay, Recombinant, Western Blot, Inhibition

    Tumor necrosis factor (TNF)-induced neurotoxicity and caspase activation in N27 dopaminergic neuronal cells. (A) Visualization of TNF toxicity by Sytox fluorescence cytotoxicity assay. N27 dopaminergic cells treated with TNF (30 ng/ml), or pretreated with etanercept (5 μg/ml) for 30 minutes and were processed for Sytox green imaging at 16 h. Increased green fluorescence is evident with TNF treatment, indicating significant neurotoxicity which was also evident in the phase contrast images. (B) Sytox fluorescence was quantified by measuring the fluorescence intensity as relative fluorescence units (RFU) using a plate reader. (C) Activation of caspase-8 by TNF treatment. Lysates from N27 cells treated with TNF (30 ng/ml) for 3 h were probed using a rabbit polyclonal antibody to caspase-8 that detects the procaspase and the active cleaved form. TNF induced a significant increase in the active caspase-8 (p20) fragment, which was blocked by pretreatment with etanercept (5 μg/ml). (D) Band intensities for cleaved caspase-8 from three blots were quantified using densitometric analysis and normalized to β-actin. (E) TNF induced activation of caspase-3. Enzymatic assays for caspase-3 activity in lysates from N27 cells treated with TNF (30 ng/ml) for 6 h or pretreated with either etanercept (5 μg/ml) or the caspase-8 inhibitor IETD-fmk (25 μM) for 30 minutes. Raw values were normalized using protein concentrations and expressed as percent control. TNF treatment induced significant activation of caspase-3, which was blocked by etanercept and the caspase-8 inhibitor IETD-fmk. (F) TNF-induced DNA fragmentation. N27 cells were treated with TNF (30 ng/ml) for 16 h or pretreated for 30 minutes with either etanercept (5 μg/ml) or the caspase-8 peptide inhibitor IETD-fmk (25 μM), and processed for the DNA fragmentation assay. Raw values were normalized using protein concentration and expressed as percent control. TNF treatment significantly increased DNA fragmentation, which was attenuated by etanercept or caspase-8 inhibition. Data represent the group mean ± SEM; n = 6 to 8 per group and experiments were repeated three times. ** ( P

    Journal: Journal of Neuroinflammation

    Article Title: Proteolytic activation of proapoptotic kinase protein kinase C? by tumor necrosis factor ? death receptor signaling in dopaminergic neurons during neuroinflammation

    doi: 10.1186/1742-2094-9-82

    Figure Lengend Snippet: Tumor necrosis factor (TNF)-induced neurotoxicity and caspase activation in N27 dopaminergic neuronal cells. (A) Visualization of TNF toxicity by Sytox fluorescence cytotoxicity assay. N27 dopaminergic cells treated with TNF (30 ng/ml), or pretreated with etanercept (5 μg/ml) for 30 minutes and were processed for Sytox green imaging at 16 h. Increased green fluorescence is evident with TNF treatment, indicating significant neurotoxicity which was also evident in the phase contrast images. (B) Sytox fluorescence was quantified by measuring the fluorescence intensity as relative fluorescence units (RFU) using a plate reader. (C) Activation of caspase-8 by TNF treatment. Lysates from N27 cells treated with TNF (30 ng/ml) for 3 h were probed using a rabbit polyclonal antibody to caspase-8 that detects the procaspase and the active cleaved form. TNF induced a significant increase in the active caspase-8 (p20) fragment, which was blocked by pretreatment with etanercept (5 μg/ml). (D) Band intensities for cleaved caspase-8 from three blots were quantified using densitometric analysis and normalized to β-actin. (E) TNF induced activation of caspase-3. Enzymatic assays for caspase-3 activity in lysates from N27 cells treated with TNF (30 ng/ml) for 6 h or pretreated with either etanercept (5 μg/ml) or the caspase-8 inhibitor IETD-fmk (25 μM) for 30 minutes. Raw values were normalized using protein concentrations and expressed as percent control. TNF treatment induced significant activation of caspase-3, which was blocked by etanercept and the caspase-8 inhibitor IETD-fmk. (F) TNF-induced DNA fragmentation. N27 cells were treated with TNF (30 ng/ml) for 16 h or pretreated for 30 minutes with either etanercept (5 μg/ml) or the caspase-8 peptide inhibitor IETD-fmk (25 μM), and processed for the DNA fragmentation assay. Raw values were normalized using protein concentration and expressed as percent control. TNF treatment significantly increased DNA fragmentation, which was attenuated by etanercept or caspase-8 inhibition. Data represent the group mean ± SEM; n = 6 to 8 per group and experiments were repeated three times. ** ( P

    Article Snippet: The results from our mechanistic studies using N27 rat dopaminergic cells indeed demonstrate for the first time that soluble TNF induces a time-dependent and dose-dependent proteolytic cleavage of PKCδ by a caspase-8 and caspase-3 signaling pathway, indicative of canonical TNF death receptor signaling via caspase-8.

    Techniques: Activation Assay, Fluorescence, Cytotoxicity Assay, Imaging, Activity Assay, DNA Fragmentation Assay, Protein Concentration, Inhibition

    Styrene oxide induced the onset of apoptosis. The subconfluence C2C12 myoblasts were treated with styrene or styrene oxide at the indicated concentrations in a growth medium for 48 h. After treatment, the nucleus was stained with Hoechst 43332 (a) and nuclear size was measured (b). The apoptosis markers in styrene oxide (c-d) or styrene (e-f) treatment were detected by Western blot with anti-caspase-9, anti-caspase-8, and anti-caspase-3 antibodies. ∗ p

    Journal: Journal of Toxicology

    Article Title: Styrene Oxide Caused Cell Cycle Arrest and Abolished Myogenic Differentiation of C2C12 Myoblasts

    doi: 10.1155/2020/1807126

    Figure Lengend Snippet: Styrene oxide induced the onset of apoptosis. The subconfluence C2C12 myoblasts were treated with styrene or styrene oxide at the indicated concentrations in a growth medium for 48 h. After treatment, the nucleus was stained with Hoechst 43332 (a) and nuclear size was measured (b). The apoptosis markers in styrene oxide (c-d) or styrene (e-f) treatment were detected by Western blot with anti-caspase-9, anti-caspase-8, and anti-caspase-3 antibodies. ∗ p

    Article Snippet: The membrane was then incubated overnight with the desired primary antibodies such as anti-myosin heavy chain (MHC), anti-caspase-3, anti-caspase-9, anti-caspase-8, and anti-tubulin.

    Techniques: Staining, Western Blot

    Expression of the p43/41‐caspase‐8 variant and its bioactivity are associated with the conversion of TRAIL‐induced autophagy to apoptosis in U937 macrophages. Cells were treated with rsTRAIL (500 ng/ml) in the presence

    Journal: Molecular Oncology

    Article Title: RIP1 modulates death receptor mediated apoptosis and autophagy in macrophages), RIP1 modulates death receptor mediated apoptosis and autophagy in macrophages

    doi: 10.1016/j.molonc.2014.12.004

    Figure Lengend Snippet: Expression of the p43/41‐caspase‐8 variant and its bioactivity are associated with the conversion of TRAIL‐induced autophagy to apoptosis in U937 macrophages. Cells were treated with rsTRAIL (500 ng/ml) in the presence

    Article Snippet: Antibodies against caspase‐8, caspase‐3 (9662), caspase‐9 (9508), phospho‐IKKα/β, IKKα, phospho‐IκBα, TRADD and FADD were purchased from Cell Signaling Technology, Inc. (Beverly, MA), antibodies against Beclin 1 (sc‐11427), Ub (sc‐8017) and IκBα (sc‐24502) from Santa Cruz Biotechnology (Santa Cruz, CA), anti‐FLIP from Alexis Biochemicals (San Diego, CA), anti‐LC3B from Sigma–Aldrich Co. (Taufkirchen, Germany), anti‐NEMO from Proteintech Group Co. (Chicago, IL), anti‐GAPDH from Kangcheng Co. (Shanghai, China) and anti‐RIP1 (610458) from BD Pharmingen (San Diego, CA).

    Techniques: Expressing, Variant Assay

    Recombinant caspase-3 and -6, but not caspase-8, cleave PPARγ. Upper panels , caspases-3 and -6 mediate cleavage of PPARγ protein and generate the 45-kDa PPARγ fragment. Isolated nuclear subcellular fractions from 3T3-L1 adipocytes

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor Necrosis Factor-? Induces Caspase-mediated Cleavage of Peroxisome Proliferator-activated Receptor ? in Adipocytes *

    doi: 10.1074/jbc.M809042200

    Figure Lengend Snippet: Recombinant caspase-3 and -6, but not caspase-8, cleave PPARγ. Upper panels , caspases-3 and -6 mediate cleavage of PPARγ protein and generate the 45-kDa PPARγ fragment. Isolated nuclear subcellular fractions from 3T3-L1 adipocytes

    Article Snippet: Rabbit anti-caspase-8 (catalog number 4927), anti-caspase-3 (catalog number 9662), and anti-cleaved-(Asp175 )-caspase-3 (catalog number 9661) polyclonal antibodies were from Cell Signaling Technologies.

    Techniques: Recombinant, Isolation

    TNFα induces nuclear translocation of active caspase-3. A , upper panels , immunoblotting of pro-caspase-8 in 3T3-L1 adipocytes cells treated or not with 50 ng/ml TNF-α plus MG132 in the absence or presence of 50 μ m Boc- d -FMK, as

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor Necrosis Factor-? Induces Caspase-mediated Cleavage of Peroxisome Proliferator-activated Receptor ? in Adipocytes *

    doi: 10.1074/jbc.M809042200

    Figure Lengend Snippet: TNFα induces nuclear translocation of active caspase-3. A , upper panels , immunoblotting of pro-caspase-8 in 3T3-L1 adipocytes cells treated or not with 50 ng/ml TNF-α plus MG132 in the absence or presence of 50 μ m Boc- d -FMK, as

    Article Snippet: Rabbit anti-caspase-8 (catalog number 4927), anti-caspase-3 (catalog number 9662), and anti-cleaved-(Asp175 )-caspase-3 (catalog number 9661) polyclonal antibodies were from Cell Signaling Technologies.

    Techniques: Translocation Assay

    3-Methyladenine enhances the fluoxetine-induced apoptosis through the death receptor pathway. (A) Cells were pre-treated with 3-MA for 1 h, followed by 10 μM fluoxetine for 24 h, and then stained with DAPI to visualize the apoptotic bodies. (B) Under the same conditions described above, cells were stained with Annexin V-DY634 and PI and analyzed by using flow cytometry. Q1=dead cells; Q2=live cells; Q3=early apoptotic cells; Q4=late apoptotic and dead cells. (C) Western blotting analysis of death receptor (DR)4, DR5, caspase 8, and caspase 3 protein expression was conducted, with β-actin used as a loading control. The blots were quantified by densitometry. The data are presented as the mean ± SEM of three independent experiments (n=3). Student’s  t -test was used to calculate statistical significance ( * p

    Journal: Biomolecules & Therapeutics

    Article Title: Fluoxetine Simultaneously Induces Both Apoptosis and Autophagy in Human Gastric Adenocarcinoma Cells

    doi: 10.4062/biomolther.2019.103

    Figure Lengend Snippet: 3-Methyladenine enhances the fluoxetine-induced apoptosis through the death receptor pathway. (A) Cells were pre-treated with 3-MA for 1 h, followed by 10 μM fluoxetine for 24 h, and then stained with DAPI to visualize the apoptotic bodies. (B) Under the same conditions described above, cells were stained with Annexin V-DY634 and PI and analyzed by using flow cytometry. Q1=dead cells; Q2=live cells; Q3=early apoptotic cells; Q4=late apoptotic and dead cells. (C) Western blotting analysis of death receptor (DR)4, DR5, caspase 8, and caspase 3 protein expression was conducted, with β-actin used as a loading control. The blots were quantified by densitometry. The data are presented as the mean ± SEM of three independent experiments (n=3). Student’s t -test was used to calculate statistical significance ( * p

    Article Snippet: For the cell viability assay, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. Anti-LC3B , anti-Atg5 , anti-BECLIN1 , anti-p62/SQSTM1 , anti-mTOR, anti-p-mTOR, anti-Akt, anti-p-Akt, anti-PARP, DR4, DR5, caspase 3, and caspase 8 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Staining, Flow Cytometry, Western Blot, Expressing

    In vivo anticancer effects of TIPE-2 (6.0 mg/kg) treatment on hepatocellular carcinoma-bearing mice. (A) Antitumor effects of TIPE-2 treatment on HepG2-bearing nude mice. (B) TIPE-2 treatment promoted apoptosis of cells within tumor tissues. (C) TIPE-2 treatment increased the expression levels of caspase-3 and caspase-8 in tumor tissues. Effects of TIPE-2 treatment on the expression levels of (D) PI3K and AKT, and (E) GRP78 and CHOP in tumor tissues. Magnification, ×40. (F) Survival rate of HepG2-bearing mice following treatment with TIPE-2 or PBS. **P

    Journal: Molecular Medicine Reports

    Article Title: TIPE-2 suppresses growth and aggressiveness of hepatocellular carcinoma cells through downregulation of the phosphoinositide 3-kinase/AKT signaling pathway

    doi: 10.3892/mmr.2018.8789

    Figure Lengend Snippet: In vivo anticancer effects of TIPE-2 (6.0 mg/kg) treatment on hepatocellular carcinoma-bearing mice. (A) Antitumor effects of TIPE-2 treatment on HepG2-bearing nude mice. (B) TIPE-2 treatment promoted apoptosis of cells within tumor tissues. (C) TIPE-2 treatment increased the expression levels of caspase-3 and caspase-8 in tumor tissues. Effects of TIPE-2 treatment on the expression levels of (D) PI3K and AKT, and (E) GRP78 and CHOP in tumor tissues. Magnification, ×40. (F) Survival rate of HepG2-bearing mice following treatment with TIPE-2 or PBS. **P

    Article Snippet: Membranes were blocked with 5% milk for 2 h at 37°C prior to incubation with the following primary antibodies overnight at 4°C: PI3K (1:1,000; cat. no. ab191606), AKT (1:1,000; cat. no. ab8805), neuroblastoma Ras viral oncogene (N-ras; 1:1,000; cat. no. ab206969), P27 (1:1,000; cat. no. ab191606), phosphorylated (p)AKT (1:1,000; cat. no. ab81283), binding immunoglobulin protein (BIP; 1:1,000; cat. no. ab108615), P53 (1:1,000; cat. no. ab1431), B-cell lymphoma 2 (Bcl-2; 1:1,000; cat. no. ab59348), cyclin D1 (1:1,000; cat. no. ab134175), cyclin-dependent kinase (CDK)1 (1:1,000; cat. no. ab131450), CDK2 (1:1,000; cat. no. ab32147), vimentin (VIM; 1:1,000; cat. no. ab92547), collagen type I (CT-I; 1:1,000; cat. no. ab34710), Slug (1:1,000; cat. no. ab27568), c-Jun N-terminal kinase (JNK; 1:1,000; cat. no. ab124956), nuclear factor (NF)-κB (1:1,000; cat. no. ab28849), nuclear factor (erythroid-derived 2)-like 2 (NRF2) (1:1,000; cat. no. ab62352), caspase-3 (1:1,000; cat. no. ab13847), caspase-8 (1:1,000; cat. no. ab25901), glucose-regulated protein 78 (GRP78; 1:1,000; cat. no. ab21685), CCAAT-enhancer-binding protein homologous protein (CHOP; 1:1,000; cat. no. ab179823), eukaryotic initiation factor 2 (eIF2α; 1:1,000; cat. no. ab32713), peIF2α (1:1,000; cat. no. ab214434) and β-actin (1:1,000; cat. no. ab827; Abcam, Shanghai, China) for 12 h at 4°C.

    Techniques: In Vivo, Mouse Assay, Expressing

    Effects of TIPE-2 (2.0 mg/ml) on hepatocellular carcinoma cell apoptosis in vitro . (A) Effects of TIPE-2 on apoptosis of HepG2 cells after 48 h, as determined by flow cytometry. Effects of TIPE-2 treatment on the expression levels of (B) caspase-3 and caspase-8, (C) P53 and Bcl-2, (D) peIF2α and eIF2α, (E) NRF2 and BIP, and (F) GRP78 and CHOP in HepG2 cells, as determined by western blotting. **P

    Journal: Molecular Medicine Reports

    Article Title: TIPE-2 suppresses growth and aggressiveness of hepatocellular carcinoma cells through downregulation of the phosphoinositide 3-kinase/AKT signaling pathway

    doi: 10.3892/mmr.2018.8789

    Figure Lengend Snippet: Effects of TIPE-2 (2.0 mg/ml) on hepatocellular carcinoma cell apoptosis in vitro . (A) Effects of TIPE-2 on apoptosis of HepG2 cells after 48 h, as determined by flow cytometry. Effects of TIPE-2 treatment on the expression levels of (B) caspase-3 and caspase-8, (C) P53 and Bcl-2, (D) peIF2α and eIF2α, (E) NRF2 and BIP, and (F) GRP78 and CHOP in HepG2 cells, as determined by western blotting. **P

    Article Snippet: Membranes were blocked with 5% milk for 2 h at 37°C prior to incubation with the following primary antibodies overnight at 4°C: PI3K (1:1,000; cat. no. ab191606), AKT (1:1,000; cat. no. ab8805), neuroblastoma Ras viral oncogene (N-ras; 1:1,000; cat. no. ab206969), P27 (1:1,000; cat. no. ab191606), phosphorylated (p)AKT (1:1,000; cat. no. ab81283), binding immunoglobulin protein (BIP; 1:1,000; cat. no. ab108615), P53 (1:1,000; cat. no. ab1431), B-cell lymphoma 2 (Bcl-2; 1:1,000; cat. no. ab59348), cyclin D1 (1:1,000; cat. no. ab134175), cyclin-dependent kinase (CDK)1 (1:1,000; cat. no. ab131450), CDK2 (1:1,000; cat. no. ab32147), vimentin (VIM; 1:1,000; cat. no. ab92547), collagen type I (CT-I; 1:1,000; cat. no. ab34710), Slug (1:1,000; cat. no. ab27568), c-Jun N-terminal kinase (JNK; 1:1,000; cat. no. ab124956), nuclear factor (NF)-κB (1:1,000; cat. no. ab28849), nuclear factor (erythroid-derived 2)-like 2 (NRF2) (1:1,000; cat. no. ab62352), caspase-3 (1:1,000; cat. no. ab13847), caspase-8 (1:1,000; cat. no. ab25901), glucose-regulated protein 78 (GRP78; 1:1,000; cat. no. ab21685), CCAAT-enhancer-binding protein homologous protein (CHOP; 1:1,000; cat. no. ab179823), eukaryotic initiation factor 2 (eIF2α; 1:1,000; cat. no. ab32713), peIF2α (1:1,000; cat. no. ab214434) and β-actin (1:1,000; cat. no. ab827; Abcam, Shanghai, China) for 12 h at 4°C.

    Techniques: In Vitro, Flow Cytometry, Cytometry, Expressing, Western Blot

    Analysis of protein expression in Hepa1c1c7 cells. The expression of proteins was quantified by flow cytometry after 24 h of treatment with PHO-S, DODAC and DODAC/PHO-S. The bar graphs show the level of expression of TRAIL - DR4 receptor ( a ); protein caspase 3 ( b ); caspase-8 protein ( c ); Free cytochrome c ( d ); protein Bax ( e ); protein Bcl-2 ( f ). Values are expressed as a mean ± SD standard deviation of three independent experiments. Level of significance * P

    Journal: BMC Pharmacology & Toxicology

    Article Title: Antiproliferative and proapoptotic effects of DODAC/synthetic phosphoethanolamine on hepatocellular carcinoma cells

    doi: 10.1186/s40360-018-0225-2

    Figure Lengend Snippet: Analysis of protein expression in Hepa1c1c7 cells. The expression of proteins was quantified by flow cytometry after 24 h of treatment with PHO-S, DODAC and DODAC/PHO-S. The bar graphs show the level of expression of TRAIL - DR4 receptor ( a ); protein caspase 3 ( b ); caspase-8 protein ( c ); Free cytochrome c ( d ); protein Bax ( e ); protein Bcl-2 ( f ). Values are expressed as a mean ± SD standard deviation of three independent experiments. Level of significance * P

    Article Snippet: In brief, the Hepa1c1c7 cells (1 × 105 cells/well, the cell density reached 80 to 90% confluence), were treated with PHO-S (0.3–2.0 mM), DODAC/PHO-S 1:1 (0.3–2.0 mM), and empty DODAC (0.3–2.0 mM), for 12 h, were washed with PBS and resuspended in FACS buffer with 2.5% paraformaldehyde for 1 h. After washing, cells were again resuspended in a primary antibody specific for the proteins anti-CD44, anti-CD90, anti-p53, anti-p21, anti-p27, anti-Bax, anti-Bcl-2, anti-caspase-3, anti-caspase-8 (Abcam, Cambridge, MA, United States); anti- cytochrome c, anti-DR4 (Santa (Cruz Biotechnology Inc., Santa Cruz, EUA) and anti-cyclin D1 (Cell Signaling Technology, Danvers, MA), at a concentration of 1 μg/ml at 4 °C, for 1 min.

    Techniques: Expressing, Flow Cytometry, Cytometry, Standard Deviation

    Images of cells transfected with M mRNA or treated with inhibitors of host gene expression in the presence of a caspase-8 inhibitor. HeLa cells were untreated (A) or transfected with yeast RNA (B) as negative controls. HeLa cells were treated with TRAIL as a positive control (C and D), transfected with M mRNA (E and F), treated with actinomycin D (G and H), or treated with DRB (I and J). Cells shown in panels D, F, H, and J were pretreated with 100 μM synthetic caspase-8 inhibitor, Z-IETD-FMK. Phase-contrast images were captured with a ×20 objective. Representative images were chosen from three separate experiments.

    Journal: Journal of Virology

    Article Title: Contrasting Effects of Matrix Protein on Apoptosis in HeLa and BHK Cells Infected with Vesicular Stomatitis Virus Are due to Inhibition of Host Gene Expression

    doi: 10.1128/JVI.77.8.4658-4669.2003

    Figure Lengend Snippet: Images of cells transfected with M mRNA or treated with inhibitors of host gene expression in the presence of a caspase-8 inhibitor. HeLa cells were untreated (A) or transfected with yeast RNA (B) as negative controls. HeLa cells were treated with TRAIL as a positive control (C and D), transfected with M mRNA (E and F), treated with actinomycin D (G and H), or treated with DRB (I and J). Cells shown in panels D, F, H, and J were pretreated with 100 μM synthetic caspase-8 inhibitor, Z-IETD-FMK. Phase-contrast images were captured with a ×20 objective. Representative images were chosen from three separate experiments.

    Article Snippet: If the caspase-8 pathway is activated by M protein and the caspase-9 pathway is activated by cross talk, then we would expect Bcl-2 to prevent the activation of caspase-9 but not caspase-8.

    Techniques: Transfection, Expressing, Positive Control

    Confocal microscopy study of the binding of Bid and caspase-8 to giant unilamellar vesicles containing cardiolipin. Trios of images (top, middle and bottom) for the same sample: two images obtained with two different detector channels of the microscope, together with an overlay image. DOPC-only (100∶0) vesicles are presented in panels a to c and DOPC/CL (90∶10) vesicles in panels d to f . Top: in a and d , protein binding to GUVs shown in green (this binding only becomes apparent when the green label accumulates at the membrane); middle: the GUV membrane was labelled with 0.05% of the hydrophobic dye DiO, as shown in ( b, c ) and in red, as shown in ( e, f ); bottom: overlay of green and red images ( c, f ). Time is indicated in minutes. The arrows indicate the decrease in GUV fluorescence following the formation of a complex between procaspase-8 and Bid Alexa488 , resulting in a non-fluorescent tBid.

    Journal: PLoS ONE

    Article Title: Caspase-8 Binding to Cardiolipin in Giant Unilamellar Vesicles Provides a Functional Docking Platform for Bid

    doi: 10.1371/journal.pone.0055250

    Figure Lengend Snippet: Confocal microscopy study of the binding of Bid and caspase-8 to giant unilamellar vesicles containing cardiolipin. Trios of images (top, middle and bottom) for the same sample: two images obtained with two different detector channels of the microscope, together with an overlay image. DOPC-only (100∶0) vesicles are presented in panels a to c and DOPC/CL (90∶10) vesicles in panels d to f . Top: in a and d , protein binding to GUVs shown in green (this binding only becomes apparent when the green label accumulates at the membrane); middle: the GUV membrane was labelled with 0.05% of the hydrophobic dye DiO, as shown in ( b, c ) and in red, as shown in ( e, f ); bottom: overlay of green and red images ( c, f ). Time is indicated in minutes. The arrows indicate the decrease in GUV fluorescence following the formation of a complex between procaspase-8 and Bid Alexa488 , resulting in a non-fluorescent tBid.

    Article Snippet: Results We tested for direct interaction between caspase-8 and cardiolipin, by incubating liposomes with the same lipid composition as the mitochondrial contact site prepared as previously described with in vitro -translated caspase-8 (p55).

    Techniques: Confocal Microscopy, Binding Assay, Microscopy, Protein Binding, Fluorescence

    Flow cytometric analysis of the interaction between CL-GUVs and caspase-8-Bid. ( a–b ) Short-term effects (20 time points at 20 s intervals, total 6.66 min) of successive additions of procaspase-8 or Bid to GUVs-CL. ( a–b ) Each product (caspase-8 or Bid) was added progressively, at 1.66-minute intervals, as shown in the recording, and the mean fluorescence of the vesicles was then measured. ( a ) Caspase-8 (Casp8) was added before Bid whereas, in ( b ), caspase-8 was added after three successive additions of Bid (10 nM, 40 nM and 60 nM). Even shortly after additions, the enzymatic system was functional, provided that caspase-8 bound to the giant unilamelar liposomes (GUVs). ( c ) the upper histogram, in black, corresponds to ( a ), and the lower histogram, in red, corresponds to ( b ); the occurrence of vesicles with a higher side scatter (SSC), due to procaspase-8/Bid cleavage activity, was recorded and is plotted as a percentage (%) of the total vesicle population. ( d ) The intensity of Bid-Alexa 647 fluorescence associated with GUVs is shown as a function of procaspase-8 addition and time, for GUVs with (closed circles) or without (open circles) CL.

    Journal: PLoS ONE

    Article Title: Caspase-8 Binding to Cardiolipin in Giant Unilamellar Vesicles Provides a Functional Docking Platform for Bid

    doi: 10.1371/journal.pone.0055250

    Figure Lengend Snippet: Flow cytometric analysis of the interaction between CL-GUVs and caspase-8-Bid. ( a–b ) Short-term effects (20 time points at 20 s intervals, total 6.66 min) of successive additions of procaspase-8 or Bid to GUVs-CL. ( a–b ) Each product (caspase-8 or Bid) was added progressively, at 1.66-minute intervals, as shown in the recording, and the mean fluorescence of the vesicles was then measured. ( a ) Caspase-8 (Casp8) was added before Bid whereas, in ( b ), caspase-8 was added after three successive additions of Bid (10 nM, 40 nM and 60 nM). Even shortly after additions, the enzymatic system was functional, provided that caspase-8 bound to the giant unilamelar liposomes (GUVs). ( c ) the upper histogram, in black, corresponds to ( a ), and the lower histogram, in red, corresponds to ( b ); the occurrence of vesicles with a higher side scatter (SSC), due to procaspase-8/Bid cleavage activity, was recorded and is plotted as a percentage (%) of the total vesicle population. ( d ) The intensity of Bid-Alexa 647 fluorescence associated with GUVs is shown as a function of procaspase-8 addition and time, for GUVs with (closed circles) or without (open circles) CL.

    Article Snippet: Results We tested for direct interaction between caspase-8 and cardiolipin, by incubating liposomes with the same lipid composition as the mitochondrial contact site prepared as previously described with in vitro -translated caspase-8 (p55).

    Techniques: Flow Cytometry, Fluorescence, Functional Assay, Activity Assay

    Binding of Bid and caspase-8 to CL-containing large unilamellar liposomes (LUVs). ( a ) Schematic diagram of caspase-8 autoprocessing during Fas-mediated apoptosis. Upon dimerisation, procaspase-8 (p55) is initially cleaved between its two active subunits, p18 and p10, to generate the p43/p10 heterodimer; p43 is then cleaved between the death effector domain (DED) and the p18 subunit, to produce the fully active p18/p10 form. ( b ) Western blot analysis of caspase-8 binding to the “contact site mimetic” liposomes or similar liposomes without CL, in which the CL was replaced with PE (22%) ( c ) Caspase-8 binding, as detected by caspACE FITC-VAD-fmk binding to the active site, to liposomes of various compositions (monolipid liposomes made from PA, PC, PE, PI, PG or cholesterol, and mixed liposomes composed of DOPC+CL, DOPC+PE, DOPC+CL+PE at various molar ratios, contact site mimetic liposomes; for details see materials and methods). ( d ) Flow cytometric analysis of CL + and DOPC-only liposomes in the presence or absence of Bid Alexa488 . The black spectrum correspond to control vesicles whereas the red spectrum correspond to the vesicles plus Bid Alexa488 . The blue spectrum results from an alkaline wash of the CL + liposomes. The alkaline wash involved centrifugation of liposomes and resuspending them in 0.1 M Na 2 CO 3 , pH 11.5. The liposomes were then analysed directly by flow cytometry. Fm: fluorescence mean value, in arbitrary units (a.u.).

    Journal: PLoS ONE

    Article Title: Caspase-8 Binding to Cardiolipin in Giant Unilamellar Vesicles Provides a Functional Docking Platform for Bid

    doi: 10.1371/journal.pone.0055250

    Figure Lengend Snippet: Binding of Bid and caspase-8 to CL-containing large unilamellar liposomes (LUVs). ( a ) Schematic diagram of caspase-8 autoprocessing during Fas-mediated apoptosis. Upon dimerisation, procaspase-8 (p55) is initially cleaved between its two active subunits, p18 and p10, to generate the p43/p10 heterodimer; p43 is then cleaved between the death effector domain (DED) and the p18 subunit, to produce the fully active p18/p10 form. ( b ) Western blot analysis of caspase-8 binding to the “contact site mimetic” liposomes or similar liposomes without CL, in which the CL was replaced with PE (22%) ( c ) Caspase-8 binding, as detected by caspACE FITC-VAD-fmk binding to the active site, to liposomes of various compositions (monolipid liposomes made from PA, PC, PE, PI, PG or cholesterol, and mixed liposomes composed of DOPC+CL, DOPC+PE, DOPC+CL+PE at various molar ratios, contact site mimetic liposomes; for details see materials and methods). ( d ) Flow cytometric analysis of CL + and DOPC-only liposomes in the presence or absence of Bid Alexa488 . The black spectrum correspond to control vesicles whereas the red spectrum correspond to the vesicles plus Bid Alexa488 . The blue spectrum results from an alkaline wash of the CL + liposomes. The alkaline wash involved centrifugation of liposomes and resuspending them in 0.1 M Na 2 CO 3 , pH 11.5. The liposomes were then analysed directly by flow cytometry. Fm: fluorescence mean value, in arbitrary units (a.u.).

    Article Snippet: Results We tested for direct interaction between caspase-8 and cardiolipin, by incubating liposomes with the same lipid composition as the mitochondrial contact site prepared as previously described with in vitro -translated caspase-8 (p55).

    Techniques: Binding Assay, Western Blot, Flow Cytometry, Centrifugation, Cytometry, Fluorescence

    Analysis of the effects of caspase-8, Bid and tBid on the Laurdan fluorescence of CL + and CL − liposomes. Generalised polarisation (GP, arbitrary units, a.u.) determined from Laurdan fluorescence measurements. GP values are reported for the various preparations, as described in the materials and methods.

    Journal: PLoS ONE

    Article Title: Caspase-8 Binding to Cardiolipin in Giant Unilamellar Vesicles Provides a Functional Docking Platform for Bid

    doi: 10.1371/journal.pone.0055250

    Figure Lengend Snippet: Analysis of the effects of caspase-8, Bid and tBid on the Laurdan fluorescence of CL + and CL − liposomes. Generalised polarisation (GP, arbitrary units, a.u.) determined from Laurdan fluorescence measurements. GP values are reported for the various preparations, as described in the materials and methods.

    Article Snippet: Results We tested for direct interaction between caspase-8 and cardiolipin, by incubating liposomes with the same lipid composition as the mitochondrial contact site prepared as previously described with in vitro -translated caspase-8 (p55).

    Techniques: Fluorescence

    Determination of the micromechanical properties of giant unilamellar vesicles (GUVs) by microaspiration. (a) Video micrograph of a vesicle aspirated in a glass suction capillary. The principal variables for the determination of the area expansion modulus are indicated: R V : vesicle radius, p in and p out : pressure inside and outside the vesicle, ΔL: length of membrane meniscus inside a glass pipette of internal radius R p . Excess membrane tension τ is created by suction such that Δp≠0. (b and c) Histograms of the micromechanical quantities measured in the test system under various experimental conditions. (b) K s : expansion modulus (mN/m); (c) τ r : tensile breaking strength (mN/m). Caspase-8 was added to a final concentration of 290 nM, tBid to 30 nM and Bid to 50 nM. Fisher’s test were used for statistical analyses of differences for both K s and τ r measurements (**, p

    Journal: PLoS ONE

    Article Title: Caspase-8 Binding to Cardiolipin in Giant Unilamellar Vesicles Provides a Functional Docking Platform for Bid

    doi: 10.1371/journal.pone.0055250

    Figure Lengend Snippet: Determination of the micromechanical properties of giant unilamellar vesicles (GUVs) by microaspiration. (a) Video micrograph of a vesicle aspirated in a glass suction capillary. The principal variables for the determination of the area expansion modulus are indicated: R V : vesicle radius, p in and p out : pressure inside and outside the vesicle, ΔL: length of membrane meniscus inside a glass pipette of internal radius R p . Excess membrane tension τ is created by suction such that Δp≠0. (b and c) Histograms of the micromechanical quantities measured in the test system under various experimental conditions. (b) K s : expansion modulus (mN/m); (c) τ r : tensile breaking strength (mN/m). Caspase-8 was added to a final concentration of 290 nM, tBid to 30 nM and Bid to 50 nM. Fisher’s test were used for statistical analyses of differences for both K s and τ r measurements (**, p

    Article Snippet: Results We tested for direct interaction between caspase-8 and cardiolipin, by incubating liposomes with the same lipid composition as the mitochondrial contact site prepared as previously described with in vitro -translated caspase-8 (p55).

    Techniques: Transferring, Concentration Assay

    Localised production of active, cleaved BID on cardiolipin platforms that serve for the assembly of active caspase-8 and in the GUV “mimicking system”. (a) The diagram depicts the sequence of events in cells of type II according to Gonzalvez et al. [25] . The CL (red heads)/caspase-8 platform at the contact sites between inner and outer mitochondrial membranes (enriched in CL) binds BID resulting in the production of the active truncated, C-termimal part of BID (tcBID). This in turn causes CL induced perturbations of the membrane curvature, BAK/BAX oligomerization and cytochrome c release. (b) Schematic representation of the reconstituted functional platform on giant unilamellar vesicles containing CL with the p 18 /p 10 . DD, death domain; DED, death effector domain; p10 and p18 form the catalytic core of the caspase. The p43/p10 caspase-8 isoform comprises two DEDs, one p10 domain and one p18 domain. IMM, inner mitochondrial membrane; IMS, inter membrane space; OM, outer mitochondrial membrane. Red dots in the intermembrane espace, cytochrome c and the violet head correspond to the cardiolipin at the contact sites between outer and inner membrane.

    Journal: PLoS ONE

    Article Title: Caspase-8 Binding to Cardiolipin in Giant Unilamellar Vesicles Provides a Functional Docking Platform for Bid

    doi: 10.1371/journal.pone.0055250

    Figure Lengend Snippet: Localised production of active, cleaved BID on cardiolipin platforms that serve for the assembly of active caspase-8 and in the GUV “mimicking system”. (a) The diagram depicts the sequence of events in cells of type II according to Gonzalvez et al. [25] . The CL (red heads)/caspase-8 platform at the contact sites between inner and outer mitochondrial membranes (enriched in CL) binds BID resulting in the production of the active truncated, C-termimal part of BID (tcBID). This in turn causes CL induced perturbations of the membrane curvature, BAK/BAX oligomerization and cytochrome c release. (b) Schematic representation of the reconstituted functional platform on giant unilamellar vesicles containing CL with the p 18 /p 10 . DD, death domain; DED, death effector domain; p10 and p18 form the catalytic core of the caspase. The p43/p10 caspase-8 isoform comprises two DEDs, one p10 domain and one p18 domain. IMM, inner mitochondrial membrane; IMS, inter membrane space; OM, outer mitochondrial membrane. Red dots in the intermembrane espace, cytochrome c and the violet head correspond to the cardiolipin at the contact sites between outer and inner membrane.

    Article Snippet: Results We tested for direct interaction between caspase-8 and cardiolipin, by incubating liposomes with the same lipid composition as the mitochondrial contact site prepared as previously described with in vitro -translated caspase-8 (p55).

    Techniques: Sequencing, Functional Assay

    Increased RIP1/Caspase 8 containing complexes in SMCs exposing to primed RAWs and aneurysmal tissues. ( A ) SMCs were exposed to MCP-1 primed RAWs or naïve RAWs for 3 days. Left: RIP1/Caspase 8 containing complex formation was examined by in situ proximity ligation assay (PLA). Middle: co-immunostaining for SMCs (SM-αA, green) and RIP1/Caspase 8 containing complex (red spots). Nuclei (DAPI, blue). Right: magnified view of the boxed areas in the middle panel. Scale bar, 100 μm. ( B ) Increased RIP1/Caspase 8 containing complex in elastase-treated aortas. Arteries were harvested 3 days after aneurysm induction by elastase infusion. Heat-inactivated elastase served as a control. Nuclei (DAPI, blue), SMCs (SM-αA, green), RIP1/Caspase 8 containing complex (red spots). Higher magnified views of highlighted regions were shown on the right. L indicates lumen. Scale bar, 50 μm.

    Journal: PLoS ONE

    Article Title: Monocyte Chemoattractant Protein-1 (MCP-1) Regulates Macrophage Cytotoxicity in Abdominal Aortic Aneurysm

    doi: 10.1371/journal.pone.0092053

    Figure Lengend Snippet: Increased RIP1/Caspase 8 containing complexes in SMCs exposing to primed RAWs and aneurysmal tissues. ( A ) SMCs were exposed to MCP-1 primed RAWs or naïve RAWs for 3 days. Left: RIP1/Caspase 8 containing complex formation was examined by in situ proximity ligation assay (PLA). Middle: co-immunostaining for SMCs (SM-αA, green) and RIP1/Caspase 8 containing complex (red spots). Nuclei (DAPI, blue). Right: magnified view of the boxed areas in the middle panel. Scale bar, 100 μm. ( B ) Increased RIP1/Caspase 8 containing complex in elastase-treated aortas. Arteries were harvested 3 days after aneurysm induction by elastase infusion. Heat-inactivated elastase served as a control. Nuclei (DAPI, blue), SMCs (SM-αA, green), RIP1/Caspase 8 containing complex (red spots). Higher magnified views of highlighted regions were shown on the right. L indicates lumen. Scale bar, 50 μm.

    Article Snippet: Primary antibodies used include anti-cleaved caspase-8, anti-cleaved caspase-9, anti-cleaved caspase-3, anti-cleaved PARP, anti-β-actin (Cell Signaling Technologies, Danvers, MA), anti-Fas Ligand, anti-alpha smooth muscle Actin antibody (Abcam, Cambridge, MA), anti-CD68 (AbD Serotec, Raleigh, NC), Rat Anti-Mouse Fas Ligand Monoclonal Antibody for neutralization, Rat IgG1 Isotype Control (R & D Systems, Minneapolis, MN), Anti-Mouse Fas Ligand FITC, Armenian Hamster IgG Isotype Control FITC (eBioscience, San Diego, CA).

    Techniques: In Situ, Proximity Ligation Assay, Immunostaining

    Receptor interacting protein-1 (RIP-1) underlies SMC apoptosis in the co-culture. ( A ) Inhibition of RIP1 with necrostatin-1 (nec-1, 40 μM) profoundly diminished apoptosis in the co-culture. ( B ) Efficiency of siRNA-mediated knockdown of RIP1 was examined by real-time PCR analysis at 24 hours and 72 hours after siRNA transfection to SMC. ( C ) SiRNA-mediated knockdown of RIP1 in SMCs prior to exposing SMCs to macrophages significantly attenuated apoptosis in the co-culture. Apoptosis was evaluated by Western blot analysis of cleaved caspase-8 and -3. Data are mean±SEM. n = 3∼4, # p

    Journal: PLoS ONE

    Article Title: Monocyte Chemoattractant Protein-1 (MCP-1) Regulates Macrophage Cytotoxicity in Abdominal Aortic Aneurysm

    doi: 10.1371/journal.pone.0092053

    Figure Lengend Snippet: Receptor interacting protein-1 (RIP-1) underlies SMC apoptosis in the co-culture. ( A ) Inhibition of RIP1 with necrostatin-1 (nec-1, 40 μM) profoundly diminished apoptosis in the co-culture. ( B ) Efficiency of siRNA-mediated knockdown of RIP1 was examined by real-time PCR analysis at 24 hours and 72 hours after siRNA transfection to SMC. ( C ) SiRNA-mediated knockdown of RIP1 in SMCs prior to exposing SMCs to macrophages significantly attenuated apoptosis in the co-culture. Apoptosis was evaluated by Western blot analysis of cleaved caspase-8 and -3. Data are mean±SEM. n = 3∼4, # p

    Article Snippet: Primary antibodies used include anti-cleaved caspase-8, anti-cleaved caspase-9, anti-cleaved caspase-3, anti-cleaved PARP, anti-β-actin (Cell Signaling Technologies, Danvers, MA), anti-Fas Ligand, anti-alpha smooth muscle Actin antibody (Abcam, Cambridge, MA), anti-CD68 (AbD Serotec, Raleigh, NC), Rat Anti-Mouse Fas Ligand Monoclonal Antibody for neutralization, Rat IgG1 Isotype Control (R & D Systems, Minneapolis, MN), Anti-Mouse Fas Ligand FITC, Armenian Hamster IgG Isotype Control FITC (eBioscience, San Diego, CA).

    Techniques: Co-Culture Assay, Inhibition, Real-time Polymerase Chain Reaction, Transfection, Western Blot

    Activation of Caspase 8/Caspase 3-mediated cell death pathway in the co-culture. ( A ) Representative immunoblots of cleaved caspase-8, -9, -3, and –PARP in SMCs, RAWs, and co-cultures as indicated. RAWs were primed by treatment of MCP-1 (100 ng/ml) for 24 h prior to co-culture. Cells were harvested after 3 days of co-culture or individual culture. ( B ) Quantifications of Western blots for cleaved caspase-8, -9, -3, and -PARP from SMCs co-cultured with naïve or MCP-1 (100 ng/ml, 24 h) primed RAWs. Data are mean±SEM. n = 3∼6, # p

    Journal: PLoS ONE

    Article Title: Monocyte Chemoattractant Protein-1 (MCP-1) Regulates Macrophage Cytotoxicity in Abdominal Aortic Aneurysm

    doi: 10.1371/journal.pone.0092053

    Figure Lengend Snippet: Activation of Caspase 8/Caspase 3-mediated cell death pathway in the co-culture. ( A ) Representative immunoblots of cleaved caspase-8, -9, -3, and –PARP in SMCs, RAWs, and co-cultures as indicated. RAWs were primed by treatment of MCP-1 (100 ng/ml) for 24 h prior to co-culture. Cells were harvested after 3 days of co-culture or individual culture. ( B ) Quantifications of Western blots for cleaved caspase-8, -9, -3, and -PARP from SMCs co-cultured with naïve or MCP-1 (100 ng/ml, 24 h) primed RAWs. Data are mean±SEM. n = 3∼6, # p

    Article Snippet: Primary antibodies used include anti-cleaved caspase-8, anti-cleaved caspase-9, anti-cleaved caspase-3, anti-cleaved PARP, anti-β-actin (Cell Signaling Technologies, Danvers, MA), anti-Fas Ligand, anti-alpha smooth muscle Actin antibody (Abcam, Cambridge, MA), anti-CD68 (AbD Serotec, Raleigh, NC), Rat Anti-Mouse Fas Ligand Monoclonal Antibody for neutralization, Rat IgG1 Isotype Control (R & D Systems, Minneapolis, MN), Anti-Mouse Fas Ligand FITC, Armenian Hamster IgG Isotype Control FITC (eBioscience, San Diego, CA).

    Techniques: Activation Assay, Co-Culture Assay, Western Blot, Cell Culture

    The expression and activation of apoptosis-associated molecules in GBM cells treated with BP. Whole cell lysates (20 µg/lane) were analyzed with western blotting using specific antibodies to: (a) p53, phospho-p53 and phospho-RB; (b) p16, p21, p27, cdk2, cdk4, cdk6, cyclin D1 and cyclin E; (c) Bax, AIF and caspase 9; (d) Fas, Fas-L, caspase 8 and caspase 3. Lower panel: Relation to control in (a) to (d) is relative to untreated control cells. Positive control of RG2 cells is DBTRG-05 MG cells with BP treatment for 3 h. UD, protein undetectable in this western blot system: ND, not detected.

    Journal: Journal of Neurochemistry

    Article Title: The natural compound n-butylidenephthalide derived from Angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo 3

    doi: 10.1111/j.1471-4159.2006.04151.x

    Figure Lengend Snippet: The expression and activation of apoptosis-associated molecules in GBM cells treated with BP. Whole cell lysates (20 µg/lane) were analyzed with western blotting using specific antibodies to: (a) p53, phospho-p53 and phospho-RB; (b) p16, p21, p27, cdk2, cdk4, cdk6, cyclin D1 and cyclin E; (c) Bax, AIF and caspase 9; (d) Fas, Fas-L, caspase 8 and caspase 3. Lower panel: Relation to control in (a) to (d) is relative to untreated control cells. Positive control of RG2 cells is DBTRG-05 MG cells with BP treatment for 3 h. UD, protein undetectable in this western blot system: ND, not detected.

    Article Snippet: The antibodies included anti-Fas, anti-Fas-L, anti-caspase 3, anti-caspase 8, anti-caspase 9, anti-Bax, anti-AIF, anti-p16, anti-p21, anti-p27, anti-p53, anti-cdk2, anti-cdk4, anti-cdk6, anti-cyclin D1, anti-cyclin E, anti-actin (1/200 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); anti-phospho-p53 (Ser15; 1/2000 dilution), and anti-phospho-RB (Ser795; 1/2000 dilution; Cell Signaling Technology, Beverly, MA, USA).The immobilized primary antigen-antibody complex was detected with the respective horseradish peroxidase-conjugated anti-mouse, anti-rabbit or anti-goat IgG secondary antibodies (1/1000 dilution; Santa Cruz Biotechnology Inc.) for 1 h at 25°C, then visualized with an enhanced chemiluminescence (ECL) Plus chemiluminescence system (Amersham, Arlington Heights, IL, USA).

    Techniques: Expressing, Activation Assay, Western Blot, Positive Control

    Effect of EG on the expression of apoptosis-associated proteins in HL-60 cells. HL-60 cells (5 × 10 5 cells) were treated with 50 μM or 75 μM EG for 6 h, 12 h, or 24 h. Cell lysates were resolved by SDS-PAGE and subjected to western blotting. ( A ) Bcl-2, Bax, and tBid. Con: Control, Cyto: cytosol, Mito: mitochondria; ( B ) Caspase-8, caspase-9, caspase-3, apoptosis-inducing factor (AIF), and Endo G; ( C ) Cytochrome c . These results presented are representative of data obtained from three independent experiments carried out in triplicate. These results presented are representative of data obtained from three independent experiments carried out in triplicate.

    Journal: International Journal of Molecular Sciences

    Article Title: Ethyl Gallate Induces Apoptosis of HL-60 Cells by Promoting the Expression of Caspases-8, -9, -3, Apoptosis-Inducing Factor and Endonuclease G

    doi: 10.3390/ijms130911912

    Figure Lengend Snippet: Effect of EG on the expression of apoptosis-associated proteins in HL-60 cells. HL-60 cells (5 × 10 5 cells) were treated with 50 μM or 75 μM EG for 6 h, 12 h, or 24 h. Cell lysates were resolved by SDS-PAGE and subjected to western blotting. ( A ) Bcl-2, Bax, and tBid. Con: Control, Cyto: cytosol, Mito: mitochondria; ( B ) Caspase-8, caspase-9, caspase-3, apoptosis-inducing factor (AIF), and Endo G; ( C ) Cytochrome c . These results presented are representative of data obtained from three independent experiments carried out in triplicate. These results presented are representative of data obtained from three independent experiments carried out in triplicate.

    Article Snippet: Caspase-3 (Cell Signaling Technology; Danvers, MA, USA); caspase-8, caspase-9, and Endo G (Enzo Life Sciences; Farmingdale, NY, USA); AIF (Bethyl Laboratories; Montgomery, TX, USA); B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Bcl-2-interacting domain (Bid) (Santa Cruz Biotechnology; Santa Cruz, CA, USA); cytochrome c (BioVision; Conesa, Buenos Aires, Argentina); and β-actin (Sigma-Aldrich; St. Louis, MO, USA).

    Techniques: Expressing, SDS Page, Western Blot

    Representative photomicrographs at 10× magnification with 20× magnification inset panel and scale bar representing 100 μm. a Caspase-3 high expression; b caspase-3 low expression; c caspase-8 high expression; d caspase-8 low expression

    Journal: Apoptosis

    Article Title: Caspase-3 and caspase-8 expression in breast cancer: caspase-3 is associated with survival

    doi: 10.1007/s10495-016-1323-5

    Figure Lengend Snippet: Representative photomicrographs at 10× magnification with 20× magnification inset panel and scale bar representing 100 μm. a Caspase-3 high expression; b caspase-3 low expression; c caspase-8 high expression; d caspase-8 low expression

    Article Snippet: A few TMA cores were not assessed due to insufficient tumour or the core being missing, a total number of 1421 cases for caspase-3, and 1402 cases for caspase-8 were assessed.

    Techniques: Expressing

    Kaplan–Meier survival curves for breast cancer specific-survival based upon caspase-3/caspase-8 expression, showing as different subgroups. Panel A caspase-3 expression with HER2 negative and HER2 positive diseases. Panel B caspase-3 expression with non-triple negative and triple-negative diseases. Panel C caspase-3 expression with non-basal like diseases and basal-like diseases. Panel D caspase-8 expression with HER2 negative and HER2 positive diseases. Panel E caspase-8 expression with non-triple negative and triple-negative diseases. Panel F caspase-8 expression with non-basal like diseases and basal-like diseases

    Journal: Apoptosis

    Article Title: Caspase-3 and caspase-8 expression in breast cancer: caspase-3 is associated with survival

    doi: 10.1007/s10495-016-1323-5

    Figure Lengend Snippet: Kaplan–Meier survival curves for breast cancer specific-survival based upon caspase-3/caspase-8 expression, showing as different subgroups. Panel A caspase-3 expression with HER2 negative and HER2 positive diseases. Panel B caspase-3 expression with non-triple negative and triple-negative diseases. Panel C caspase-3 expression with non-basal like diseases and basal-like diseases. Panel D caspase-8 expression with HER2 negative and HER2 positive diseases. Panel E caspase-8 expression with non-triple negative and triple-negative diseases. Panel F caspase-8 expression with non-basal like diseases and basal-like diseases

    Article Snippet: A few TMA cores were not assessed due to insufficient tumour or the core being missing, a total number of 1421 cases for caspase-3, and 1402 cases for caspase-8 were assessed.

    Techniques: Expressing

    Kaplan–Meier survival curve analysis showing caspase-3 ( panel A ), caspase-8 ( panel B ) and combinatorial caspase-3 and -8 expression ( panel C ), related breast cancer-specific survival; significance was determined using the log rank test. Numbers below the graph show patients at risk at the specified months. Panel A low caspase-3 ( a ) and high caspase-3 ( b ) expression. Panel B low caspase-8 ( a ) and high caspase-8 ( b ) expression. Panel C low caspase-3/-8 ( a ), high caspase-3/low caspase-8 ( b ), low caspase-3/high caspase-8 ( c ), high caspase-3/high caspase-8 ( d )

    Journal: Apoptosis

    Article Title: Caspase-3 and caspase-8 expression in breast cancer: caspase-3 is associated with survival

    doi: 10.1007/s10495-016-1323-5

    Figure Lengend Snippet: Kaplan–Meier survival curve analysis showing caspase-3 ( panel A ), caspase-8 ( panel B ) and combinatorial caspase-3 and -8 expression ( panel C ), related breast cancer-specific survival; significance was determined using the log rank test. Numbers below the graph show patients at risk at the specified months. Panel A low caspase-3 ( a ) and high caspase-3 ( b ) expression. Panel B low caspase-8 ( a ) and high caspase-8 ( b ) expression. Panel C low caspase-3/-8 ( a ), high caspase-3/low caspase-8 ( b ), low caspase-3/high caspase-8 ( c ), high caspase-3/high caspase-8 ( d )

    Article Snippet: A few TMA cores were not assessed due to insufficient tumour or the core being missing, a total number of 1421 cases for caspase-3, and 1402 cases for caspase-8 were assessed.

    Techniques: Expressing

    Antibody validation for anti-caspase-3 ( panel A ) and anti-caspase-8 ( panel B ). A single specific band, of 35 kDa, was obtained for caspase-3, and a single specific band, of 62 kDa, obtained for caspase-8. Expression, as shown, was assessed across a range of breast cancer cell lines, representing different phenotypes (MDA-MB-231, MDA-MB-435, T-47D, MCF-7 and SKBR3), human umbilical vein endothelial cells (HUVEC), fibroblasts (MRC-5), cervical cancer (HeLa) and/or colorectal cancer cells (SW480’s). Expression of caspase-3 was absent, as expected, from MCF-7’s

    Journal: Apoptosis

    Article Title: Caspase-3 and caspase-8 expression in breast cancer: caspase-3 is associated with survival

    doi: 10.1007/s10495-016-1323-5

    Figure Lengend Snippet: Antibody validation for anti-caspase-3 ( panel A ) and anti-caspase-8 ( panel B ). A single specific band, of 35 kDa, was obtained for caspase-3, and a single specific band, of 62 kDa, obtained for caspase-8. Expression, as shown, was assessed across a range of breast cancer cell lines, representing different phenotypes (MDA-MB-231, MDA-MB-435, T-47D, MCF-7 and SKBR3), human umbilical vein endothelial cells (HUVEC), fibroblasts (MRC-5), cervical cancer (HeLa) and/or colorectal cancer cells (SW480’s). Expression of caspase-3 was absent, as expected, from MCF-7’s

    Article Snippet: A few TMA cores were not assessed due to insufficient tumour or the core being missing, a total number of 1421 cases for caspase-3, and 1402 cases for caspase-8 were assessed.

    Techniques: Expressing, Multiple Displacement Amplification

    Effects of caspase-8 downregulation in skin maturation. A. Immunohistochemistry analysis performed in skin from wildtype and caspase-8 knock out mice. Caspase-8 DEDs are stained in green, keratin 5 in red and nuclei in blue. Brackets denote expanded epidermis in the caspase-8 knockout mice. B. Microtubule staining (red channel) in skin from wild type and caspase-8 knockout mice. Insets show magnified views of the microtubule patterning in wild type and caspase-8 knockout mice. C. Left, Confocal images of HaCat undifferentiated and differentiated cells stained for caspase-8 DEDs (green channel) and microtubules (red channel). Yellow arrows indicate centrosomes. Right, Immunoblot analysis of caspase-8 expression in undifferentiated or differentiated keratinocytes. Lysates were probed with antibody recognizing the DEDs of caspase-8.

    Journal: PLoS ONE

    Article Title: The Death Effector Domains of Caspase-8 Induce Terminal Differentiation

    doi: 10.1371/journal.pone.0007879

    Figure Lengend Snippet: Effects of caspase-8 downregulation in skin maturation. A. Immunohistochemistry analysis performed in skin from wildtype and caspase-8 knock out mice. Caspase-8 DEDs are stained in green, keratin 5 in red and nuclei in blue. Brackets denote expanded epidermis in the caspase-8 knockout mice. B. Microtubule staining (red channel) in skin from wild type and caspase-8 knockout mice. Insets show magnified views of the microtubule patterning in wild type and caspase-8 knockout mice. C. Left, Confocal images of HaCat undifferentiated and differentiated cells stained for caspase-8 DEDs (green channel) and microtubules (red channel). Yellow arrows indicate centrosomes. Right, Immunoblot analysis of caspase-8 expression in undifferentiated or differentiated keratinocytes. Lysates were probed with antibody recognizing the DEDs of caspase-8.

    Article Snippet: Cell were stained with monoclonal antibody to amino terminus death effector domain of caspase-8 (Pharmingen and Calbiochem), p53 (Cell signaling), p21 (Santa Cruz) polyclonal antibody specific for alpha tubulin (Abcam), gamma tubulin (Abcam), pericentrin (Abcam) or lamin B (Santa Cruz).

    Techniques: Immunohistochemistry, Knock-Out, Mouse Assay, Staining, Expressing

    Caspase-8 DEDs decrease tumor burden, proliferation and induce multinucleation. A. Evaluation of tumor growth in chick embryos of NB7 neuroblastoma cells stably transfected with DED-GFP or control GFP. Data were analyzed with U-Mann-Whitney Test (*, significant differences, p≤0.05). B. The proliferation capacity of NB7-GFP or NB7 DED-GFP was monitored by direct counting of cells after 24, 48, 72 and 96 hours in culture after being seeded at 100, 000 cells/ml. C. Representative immunofluorescent images are shown, with alpha tubulin (red channel) and DNA/chromosomes (blue channel) evident in mitotic cells (scale bar = 10 µm). Microscopic quantification of aberrant mitosis. Data were analyzed with T-student Test (*, significant differences, p≤0.05; **, very significant differences, p≤0.01). D. Quantification of mitotic cells expressing GFP or DED-GFP. The distribution of mitotic cells is significantly different in these populations (P = Prophase, PM = Pro-metaphase, M = Metaphase and A/T = Anaphase/Telophase). Data were analyzed with T-student Test (*, significant differences, p≤0.05). E. Confocal microscopy indicating the presence of multinucleated cells within the DED-GFP cells, but not those expressing GFP (scale bar = 10 µm). F . Quantification of multinucleated NB7 GFP and NB7 DED-GFP cells.

    Journal: PLoS ONE

    Article Title: The Death Effector Domains of Caspase-8 Induce Terminal Differentiation

    doi: 10.1371/journal.pone.0007879

    Figure Lengend Snippet: Caspase-8 DEDs decrease tumor burden, proliferation and induce multinucleation. A. Evaluation of tumor growth in chick embryos of NB7 neuroblastoma cells stably transfected with DED-GFP or control GFP. Data were analyzed with U-Mann-Whitney Test (*, significant differences, p≤0.05). B. The proliferation capacity of NB7-GFP or NB7 DED-GFP was monitored by direct counting of cells after 24, 48, 72 and 96 hours in culture after being seeded at 100, 000 cells/ml. C. Representative immunofluorescent images are shown, with alpha tubulin (red channel) and DNA/chromosomes (blue channel) evident in mitotic cells (scale bar = 10 µm). Microscopic quantification of aberrant mitosis. Data were analyzed with T-student Test (*, significant differences, p≤0.05; **, very significant differences, p≤0.01). D. Quantification of mitotic cells expressing GFP or DED-GFP. The distribution of mitotic cells is significantly different in these populations (P = Prophase, PM = Pro-metaphase, M = Metaphase and A/T = Anaphase/Telophase). Data were analyzed with T-student Test (*, significant differences, p≤0.05). E. Confocal microscopy indicating the presence of multinucleated cells within the DED-GFP cells, but not those expressing GFP (scale bar = 10 µm). F . Quantification of multinucleated NB7 GFP and NB7 DED-GFP cells.

    Article Snippet: Cell were stained with monoclonal antibody to amino terminus death effector domain of caspase-8 (Pharmingen and Calbiochem), p53 (Cell signaling), p21 (Santa Cruz) polyclonal antibody specific for alpha tubulin (Abcam), gamma tubulin (Abcam), pericentrin (Abcam) or lamin B (Santa Cruz).

    Techniques: Stable Transfection, Transfection, MANN-WHITNEY, Expressing, Confocal Microscopy

    Caspase-8 DEDs are required for terminal differentiation of monocytes. A. Images of undifferentiated U937 monocytes (control) and differentiated macrophages (scale bar = 10 µm). Immunoblot analysis of caspase-8 expression among U937 undifferentiated monocytes (control) and differentiated macrophages. Lysates were probed with antibody recognizing the DEDs of caspase-8, with analysis of tubulin shown as a loading control. B. Monocytes were induced to differentiate by treatment with phorbol esters after infection with lentivirus encoding shRNA to caspase-8 or a control shRNA. Flow cytometry of control and caspase-8 knockdown U937 cells using CD11b expression as a reporter of differentiation. DED expression during differentiation was assessed by immunoblot analysis of these lysates with an antibody to caspase-8 DED domain (inset). C. Evaluation of U937 differentiation was performed by direct microscopic assessment using standard morphological criteria (round undifferentiated and spread differentiated). Data were analyzed with Fisher's Exact Test (*, significant difference, p≤0.05).

    Journal: PLoS ONE

    Article Title: The Death Effector Domains of Caspase-8 Induce Terminal Differentiation

    doi: 10.1371/journal.pone.0007879

    Figure Lengend Snippet: Caspase-8 DEDs are required for terminal differentiation of monocytes. A. Images of undifferentiated U937 monocytes (control) and differentiated macrophages (scale bar = 10 µm). Immunoblot analysis of caspase-8 expression among U937 undifferentiated monocytes (control) and differentiated macrophages. Lysates were probed with antibody recognizing the DEDs of caspase-8, with analysis of tubulin shown as a loading control. B. Monocytes were induced to differentiate by treatment with phorbol esters after infection with lentivirus encoding shRNA to caspase-8 or a control shRNA. Flow cytometry of control and caspase-8 knockdown U937 cells using CD11b expression as a reporter of differentiation. DED expression during differentiation was assessed by immunoblot analysis of these lysates with an antibody to caspase-8 DED domain (inset). C. Evaluation of U937 differentiation was performed by direct microscopic assessment using standard morphological criteria (round undifferentiated and spread differentiated). Data were analyzed with Fisher's Exact Test (*, significant difference, p≤0.05).

    Article Snippet: Cell were stained with monoclonal antibody to amino terminus death effector domain of caspase-8 (Pharmingen and Calbiochem), p53 (Cell signaling), p21 (Santa Cruz) polyclonal antibody specific for alpha tubulin (Abcam), gamma tubulin (Abcam), pericentrin (Abcam) or lamin B (Santa Cruz).

    Techniques: Expressing, Infection, shRNA, Flow Cytometry, Cytometry

    Caspase-8 DEDs activate p53, p21 and trigger apoptosis or cell cycle arrest. A. Bright field images of NB7 neuroblastoma cells expressing DED-GFP at passages 8 (early) and 30 (late) (scale bar = 10 µm). At earlier passages, a few multinucleated DED-expressing cells are detectable (yellow arrows). By passage 30, DED-GFP cells show high numbers of elongated or apoptotic cells. Late passage cells were stained with propidium iodide and assessed by flow cytometry to quantitate the sub-G1 apoptotic population at passage 30. B. Confocal microscopy confirmed the increase in nuclear expression of p21 and p53 (two markers of cell cycle arrest) and the cytoplasmic upregulation of the neuronal differentiation marker MAP2 in DED-GFP cells at passage 25 (scale bar = 10 µm). White arrows show nuclear accumulation of p21 and p53 (red channel).

    Journal: PLoS ONE

    Article Title: The Death Effector Domains of Caspase-8 Induce Terminal Differentiation

    doi: 10.1371/journal.pone.0007879

    Figure Lengend Snippet: Caspase-8 DEDs activate p53, p21 and trigger apoptosis or cell cycle arrest. A. Bright field images of NB7 neuroblastoma cells expressing DED-GFP at passages 8 (early) and 30 (late) (scale bar = 10 µm). At earlier passages, a few multinucleated DED-expressing cells are detectable (yellow arrows). By passage 30, DED-GFP cells show high numbers of elongated or apoptotic cells. Late passage cells were stained with propidium iodide and assessed by flow cytometry to quantitate the sub-G1 apoptotic population at passage 30. B. Confocal microscopy confirmed the increase in nuclear expression of p21 and p53 (two markers of cell cycle arrest) and the cytoplasmic upregulation of the neuronal differentiation marker MAP2 in DED-GFP cells at passage 25 (scale bar = 10 µm). White arrows show nuclear accumulation of p21 and p53 (red channel).

    Article Snippet: Cell were stained with monoclonal antibody to amino terminus death effector domain of caspase-8 (Pharmingen and Calbiochem), p53 (Cell signaling), p21 (Santa Cruz) polyclonal antibody specific for alpha tubulin (Abcam), gamma tubulin (Abcam), pericentrin (Abcam) or lamin B (Santa Cruz).

    Techniques: Expressing, Staining, Flow Cytometry, Cytometry, Confocal Microscopy, Marker

    Src interacts with PP2A/C and caspase-8, and TRAIL treatment promotes their interaction. A , Src interacts with PP2A/C and caspase-8 ( Casp8 ). 293T cells were co-transfected with pcDNA3-Src (Src), caspase-8-HA, and PP2A/C-HA. After 48 h, cells were left

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of the Src-PP2A Interaction in Tumor Necrosis Factor (TNF)-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis *

    doi: 10.1074/jbc.M113.508093

    Figure Lengend Snippet: Src interacts with PP2A/C and caspase-8, and TRAIL treatment promotes their interaction. A , Src interacts with PP2A/C and caspase-8 ( Casp8 ). 293T cells were co-transfected with pcDNA3-Src (Src), caspase-8-HA, and PP2A/C-HA. After 48 h, cells were left

    Article Snippet: Rabbit anti-PP2A/C, FADD, phosphotyrosine, phospho-Src (Tyr-527), and Src antibodies and mouse anti-caspase-8 were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Transfection

    TRAIL treatment leads to redistribution of Src, PP2A/C, and caspase-8 from non-rafts to lipid rafts. A , PP2A/C and caspase-8 ( Casp8 ) localize in non-rafts, whereas Src localizes in both non-rafts and lipid rafts in the absence of treatment. BT549 cells

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of the Src-PP2A Interaction in Tumor Necrosis Factor (TNF)-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis *

    doi: 10.1074/jbc.M113.508093

    Figure Lengend Snippet: TRAIL treatment leads to redistribution of Src, PP2A/C, and caspase-8 from non-rafts to lipid rafts. A , PP2A/C and caspase-8 ( Casp8 ) localize in non-rafts, whereas Src localizes in both non-rafts and lipid rafts in the absence of treatment. BT549 cells

    Article Snippet: Rabbit anti-PP2A/C, FADD, phosphotyrosine, phospho-Src (Tyr-527), and Src antibodies and mouse anti-caspase-8 were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques:

    Caspase-8 is phosphorylated at Tyr-380 in response to TRAIL treatment. BT549 cells (2.0 × 10 7 ) were left untreated or treated with TRAIL (100 ng/ml) for 30 min. Cell lysates were prepared, immunoprecipitated with caspase-8 ( Casp8 ) antibody, and

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of the Src-PP2A Interaction in Tumor Necrosis Factor (TNF)-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis *

    doi: 10.1074/jbc.M113.508093

    Figure Lengend Snippet: Caspase-8 is phosphorylated at Tyr-380 in response to TRAIL treatment. BT549 cells (2.0 × 10 7 ) were left untreated or treated with TRAIL (100 ng/ml) for 30 min. Cell lysates were prepared, immunoprecipitated with caspase-8 ( Casp8 ) antibody, and

    Article Snippet: Rabbit anti-PP2A/C, FADD, phosphotyrosine, phospho-Src (Tyr-527), and Src antibodies and mouse anti-caspase-8 were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques: Immunoprecipitation

    Src interacts with caspase-8 and activated Src phosphorylates caspase-8 at tyrosine 380. A , Src interacts with caspase-8 ( Casp8 ). BT549 cells (2.0 × 10 7 ) were treated with TRAIL (100 ng/ml) for 30 min. Cell lysates were prepared and subjected

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of the Src-PP2A Interaction in Tumor Necrosis Factor (TNF)-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis *

    doi: 10.1074/jbc.M113.508093

    Figure Lengend Snippet: Src interacts with caspase-8 and activated Src phosphorylates caspase-8 at tyrosine 380. A , Src interacts with caspase-8 ( Casp8 ). BT549 cells (2.0 × 10 7 ) were treated with TRAIL (100 ng/ml) for 30 min. Cell lysates were prepared and subjected

    Article Snippet: Rabbit anti-PP2A/C, FADD, phosphotyrosine, phospho-Src (Tyr-527), and Src antibodies and mouse anti-caspase-8 were obtained from Cell Signaling Technology (Danvers, MA).

    Techniques:

    Role of caspases in cell death induction by MSA/paclitaxel. A. Western blot of PARP cleavage by MSA/paclitaxel. B. Western blot of cleaved caspase-8 and -9 in the presence of MSA or paclitaxel, or the combination. C. Increase of cleaved caspase-8 and caspase-9 by MSA, paclitaxel, or combination. D. Effect of caspase-8 inhibitor or caspase-9 inhibitor on cell death induction by MSA/paclitaxel. *Statistically different (P

    Journal: Cancer biology & therapy

    Article Title: Chemotherapeutic Sensitization by Endoplasmic Reticulum Stress: Increasing the Efficacy of Taxane Against Prostate Cancer

    doi:

    Figure Lengend Snippet: Role of caspases in cell death induction by MSA/paclitaxel. A. Western blot of PARP cleavage by MSA/paclitaxel. B. Western blot of cleaved caspase-8 and -9 in the presence of MSA or paclitaxel, or the combination. C. Increase of cleaved caspase-8 and caspase-9 by MSA, paclitaxel, or combination. D. Effect of caspase-8 inhibitor or caspase-9 inhibitor on cell death induction by MSA/paclitaxel. *Statistically different (P

    Article Snippet: Caspase-8 and caspase-9 inhibitors, z-IETD-FMK and z-LEHD-FMK, were purchased from Biovision (Mountain View, CA).

    Techniques: Western Blot

    Activation of apoptosis in xenograft tumors and cancer cells with suppressed levels of NAF-1. (A) Left, immunohistochemistry (IHC) analysis using an antibody against activated caspase-3 showing a higher number of active-caspase-3-positive cells in NAF-1(−) tumors. Active-caspase-3-positive cells are marked by white arrowheads. Right, quantification of staining of active caspase-3. (B) Left, IHC analysis using an antibody against γH2AX showing a higher number of γH2AX-positive cells in NAF-1(−) tumors. Right, quantification of γH2AX staining. For A,B, cells were counted in ten high-power fields (×40) for each section obtained from five mice in each group; *** P

    Journal: Journal of Cell Science

    Article Title: Activation of apoptosis in NAF-1-deficient human epithelial breast cancer cells

    doi: 10.1242/jcs.178293

    Figure Lengend Snippet: Activation of apoptosis in xenograft tumors and cancer cells with suppressed levels of NAF-1. (A) Left, immunohistochemistry (IHC) analysis using an antibody against activated caspase-3 showing a higher number of active-caspase-3-positive cells in NAF-1(−) tumors. Active-caspase-3-positive cells are marked by white arrowheads. Right, quantification of staining of active caspase-3. (B) Left, IHC analysis using an antibody against γH2AX showing a higher number of γH2AX-positive cells in NAF-1(−) tumors. Right, quantification of γH2AX staining. For A,B, cells were counted in ten high-power fields (×40) for each section obtained from five mice in each group; *** P

    Article Snippet: Caspase-3 activity was measured using a caspase-3 colorimetric activity assay kit (Chemicon), as per the manufacturer's instructions.

    Techniques: Activation Assay, Immunohistochemistry, Staining, Mouse Assay

    Caspase 8 gene ablation provides neuroprotection in vitro in primary neuronal cultures and brain organotypic cultures. ( A–B ) 3-day-old PNC derived from embryos (E16.5) of CRE3 ( A ) and N casp8 −/− homozygous mice ( B ) were treated with recombinant murine TNFα and cycloheximide (CHX), then fixed and stained with Hoechst dye and immunostained with an antibody to NeuN followed by application of Alexa Fluor 594 conjugated anti-mouse antibody. ( C1–J4 ). Untreated (UNT C1–F4 ), STS-treated, ( G1–H4 ) and recombinant murine TNFα and CHX-treated ( I1–J4 ) 14-day-old neuronal cultures derived from CRE3 and N casp8 −/− mice were preincubated with pSIVA reagent (green) for 15 min prior to Z-fix fixation, then immunostained for MAP2 (red) and stained with DAPI (blue). To assess neuronal phenotype, the transmission light, single ( C1, D1, E4, G1, H1, H3, I1, I2, I4, J1 ), double ( C2, D2, E1, E2, F1, F3, G4, H2, H4, I3, J2 ), and triple ( E3, F2, F4, G2, G3, J3, J4 ) channel images are provided. Colocalization of neuronal marker MAP2 (red) with pSIVA signal (green) resulted in yellow color denoting degenerating neurons whose number was counted and plotted as ratio to the total number of neurons ( Figure 2B ). Single and multichannel panels confirm specificity of the double labeling. PSIVA was visualized from the early (membranous staining) to later stages (staining in the cell body and nucleus, the latter in cells with loss of plasma cell integrity) of neuronal degeneration.

    Journal: PLoS ONE

    Article Title: Neuronal Deletion of Caspase 8 Protects against Brain Injury in Mouse Models of Controlled Cortical Impact and Kainic Acid-Induced Excitotoxicity

    doi: 10.1371/journal.pone.0024341

    Figure Lengend Snippet: Caspase 8 gene ablation provides neuroprotection in vitro in primary neuronal cultures and brain organotypic cultures. ( A–B ) 3-day-old PNC derived from embryos (E16.5) of CRE3 ( A ) and N casp8 −/− homozygous mice ( B ) were treated with recombinant murine TNFα and cycloheximide (CHX), then fixed and stained with Hoechst dye and immunostained with an antibody to NeuN followed by application of Alexa Fluor 594 conjugated anti-mouse antibody. ( C1–J4 ). Untreated (UNT C1–F4 ), STS-treated, ( G1–H4 ) and recombinant murine TNFα and CHX-treated ( I1–J4 ) 14-day-old neuronal cultures derived from CRE3 and N casp8 −/− mice were preincubated with pSIVA reagent (green) for 15 min prior to Z-fix fixation, then immunostained for MAP2 (red) and stained with DAPI (blue). To assess neuronal phenotype, the transmission light, single ( C1, D1, E4, G1, H1, H3, I1, I2, I4, J1 ), double ( C2, D2, E1, E2, F1, F3, G4, H2, H4, I3, J2 ), and triple ( E3, F2, F4, G2, G3, J3, J4 ) channel images are provided. Colocalization of neuronal marker MAP2 (red) with pSIVA signal (green) resulted in yellow color denoting degenerating neurons whose number was counted and plotted as ratio to the total number of neurons ( Figure 2B ). Single and multichannel panels confirm specificity of the double labeling. PSIVA was visualized from the early (membranous staining) to later stages (staining in the cell body and nucleus, the latter in cells with loss of plasma cell integrity) of neuronal degeneration.

    Article Snippet: TRAIL is known to transmit the apoptotic signals through death receptors that directly activate caspase 8 .

    Techniques: In Vitro, Derivative Assay, Mouse Assay, Recombinant, Staining, Transmission Assay, Marker, Labeling

    Caspase 8 deficiency affects functional and histopathological outcome following TBI and facilitates learning and memory retention in aged mice. ( A ) Sensorimotor function was measured as the number of foot slips while traversing the length of the beam three times in the beam walking trial. Training was followed by probe 1 (one week interval) before CCI procedure and repeated 7 and 21 days after the procedure (probe 2 and 3). P values result from Student's t-test (* p = 0.009; ** p = 0.04). ( B ) Wire grip scores were quantified using a 5-point scale [33] to assess motor function. Tests were conducted 1 day before CCI (probe 1) and 7 (probe 2; * p = 0.03) and 21 days (probe 3) after brain trauma. ( C ) Morris water maze (MWM) performance was tested in the control (Ctrl; n = 12) and N casp8 −/− (n = 15) mice. Average latency to reach and climb the platform or enter the platform zone before and after CCI is shown (mean±SEM) (* p = 0.005; ** p = 0.001; *** p = 0.009). Beam walking ( D ) and wire grip ( E ) tests were conducted in uninjured 18 month old casp8 fl/fl (Ctrl; n = 19) and N casp8 −/− (n = 20) mice (3 testing sessions, spaced one week apart). P values result from Student's t-test (* p = 0.01). ( F ) MWM task was performed in the same aged cohort to test memory retention up to 60 days after completion of the learning sessions. Average latency to reach and climb the platform is shown (mean±SEM) (* p = 0.04). ( G–I ) Histopathological outcomes were compared between the control (Ctrl) and N casp8 −/− mice 2 h, 6 h, 24 h, 48 h, and 3 weeks after the trauma (8–10 mice per time point in each experimental group). Using Aperio scanning system, lesion ( G ) and cavity ( H ) volumes were determined by summing each lesion or cavity area multiplied by the distance between each coronal slice (mean±SEM). Plots depict average lesion volume ( G : * p = 0.03; ** p = 0.002; *** p = 0.003), cavity volume ( H : * p = 0.02; ** p = 0.03), and neuropathological scores ( I : * p = 0.002; ** p = 0.02; *** p = 0.004). ( J ) Time course for density (n/mm 2 ) of NeuN-immunopositive neurons in traumatic brain lesions is depicted for the control (Ctrl) and N casp8 −/− mice. ( K ) The NeuN immunostaining results are compared among the six experimental groups [control (Ctrl) and N casp8 −/− naïve (no surgery “-”), sham (surgery “S”), and subjected to CCI (“C”) mice] in impact lesions (CCI-treated) or corresponding regions (naïve or sham-operated) at 21 day after procedure. Density (n/mm 2 ) of NeuN-immunopositive neurons in impact lesions or matching ipsilateral regions (IL) and corresponding contralateral (CL) regions of the control (Ctrl) ( L ) and N casp8 −/− ( M ) naïve (no surgery “-”), sham (surgery “S”), and subjected to CCI (“C”) mice is presented in a time-course manner ( L, M ) or at 21 day after procedure ( N ) ( J : * p = 0.02; ** p = 0.01; *** p = 0.0004; L : * p = 0.0005; ** p

    Journal: PLoS ONE

    Article Title: Neuronal Deletion of Caspase 8 Protects against Brain Injury in Mouse Models of Controlled Cortical Impact and Kainic Acid-Induced Excitotoxicity

    doi: 10.1371/journal.pone.0024341

    Figure Lengend Snippet: Caspase 8 deficiency affects functional and histopathological outcome following TBI and facilitates learning and memory retention in aged mice. ( A ) Sensorimotor function was measured as the number of foot slips while traversing the length of the beam three times in the beam walking trial. Training was followed by probe 1 (one week interval) before CCI procedure and repeated 7 and 21 days after the procedure (probe 2 and 3). P values result from Student's t-test (* p = 0.009; ** p = 0.04). ( B ) Wire grip scores were quantified using a 5-point scale [33] to assess motor function. Tests were conducted 1 day before CCI (probe 1) and 7 (probe 2; * p = 0.03) and 21 days (probe 3) after brain trauma. ( C ) Morris water maze (MWM) performance was tested in the control (Ctrl; n = 12) and N casp8 −/− (n = 15) mice. Average latency to reach and climb the platform or enter the platform zone before and after CCI is shown (mean±SEM) (* p = 0.005; ** p = 0.001; *** p = 0.009). Beam walking ( D ) and wire grip ( E ) tests were conducted in uninjured 18 month old casp8 fl/fl (Ctrl; n = 19) and N casp8 −/− (n = 20) mice (3 testing sessions, spaced one week apart). P values result from Student's t-test (* p = 0.01). ( F ) MWM task was performed in the same aged cohort to test memory retention up to 60 days after completion of the learning sessions. Average latency to reach and climb the platform is shown (mean±SEM) (* p = 0.04). ( G–I ) Histopathological outcomes were compared between the control (Ctrl) and N casp8 −/− mice 2 h, 6 h, 24 h, 48 h, and 3 weeks after the trauma (8–10 mice per time point in each experimental group). Using Aperio scanning system, lesion ( G ) and cavity ( H ) volumes were determined by summing each lesion or cavity area multiplied by the distance between each coronal slice (mean±SEM). Plots depict average lesion volume ( G : * p = 0.03; ** p = 0.002; *** p = 0.003), cavity volume ( H : * p = 0.02; ** p = 0.03), and neuropathological scores ( I : * p = 0.002; ** p = 0.02; *** p = 0.004). ( J ) Time course for density (n/mm 2 ) of NeuN-immunopositive neurons in traumatic brain lesions is depicted for the control (Ctrl) and N casp8 −/− mice. ( K ) The NeuN immunostaining results are compared among the six experimental groups [control (Ctrl) and N casp8 −/− naïve (no surgery “-”), sham (surgery “S”), and subjected to CCI (“C”) mice] in impact lesions (CCI-treated) or corresponding regions (naïve or sham-operated) at 21 day after procedure. Density (n/mm 2 ) of NeuN-immunopositive neurons in impact lesions or matching ipsilateral regions (IL) and corresponding contralateral (CL) regions of the control (Ctrl) ( L ) and N casp8 −/− ( M ) naïve (no surgery “-”), sham (surgery “S”), and subjected to CCI (“C”) mice is presented in a time-course manner ( L, M ) or at 21 day after procedure ( N ) ( J : * p = 0.02; ** p = 0.01; *** p = 0.0004; L : * p = 0.0005; ** p

    Article Snippet: TRAIL is known to transmit the apoptotic signals through death receptors that directly activate caspase 8 .

    Techniques: Functional Assay, Mouse Assay, Immunostaining

    Neuroprotective effects of caspase 8 gene ablation demonstrated for cultured primary neurons. ( A ) The ratio of surviving NeuN-positive neurons that showed Hoechst staining profile characteristic of viable cells to the total cell number (number of Hoechst-positive nuclei) was determined after 3 and 14 days of culture for untreated (UNT), staurosporine- (STS) and TNFα/CHX-treated primary cortical neurons derived from embryos (E16.5) of CRE3 and N casp8 −/− homozygous mice. Cells were counted on ten random fluorescent images (×20). ( B ) The ratio of pSIVA-positive neurons with colocalized MAP2 expression to the total number of MAP2-positive cells was determined after 14 days of culture for UNT, STS- and TNFα/CHX-treated primary cortical neurons derived from embryos of CRE3 and N casp8 −/− mice. Cells were counted on 15 random fluorescent images (×20) from 3 experiments. ( C–I ) Bar graphs present average percentages of MAP2 ( C ), NeuN ( D ), cleaved caspase 3 ( E ), GFAP ( G ), S100 ( H ), Iba1 ( I ) immunopositive staining and mean percentage of TUNEL positive signaling ( F ) in cortices (left) and basal ganglia (right) of untreated and TRAIL-treated cultured coronal brain slices derived from CRE3, casp8 fl/fl (Casp8 line) and N casp8 −/− mice after 3 days of culture. Tables 1 and 2 provide the results of statistical analyses.

    Journal: PLoS ONE

    Article Title: Neuronal Deletion of Caspase 8 Protects against Brain Injury in Mouse Models of Controlled Cortical Impact and Kainic Acid-Induced Excitotoxicity

    doi: 10.1371/journal.pone.0024341

    Figure Lengend Snippet: Neuroprotective effects of caspase 8 gene ablation demonstrated for cultured primary neurons. ( A ) The ratio of surviving NeuN-positive neurons that showed Hoechst staining profile characteristic of viable cells to the total cell number (number of Hoechst-positive nuclei) was determined after 3 and 14 days of culture for untreated (UNT), staurosporine- (STS) and TNFα/CHX-treated primary cortical neurons derived from embryos (E16.5) of CRE3 and N casp8 −/− homozygous mice. Cells were counted on ten random fluorescent images (×20). ( B ) The ratio of pSIVA-positive neurons with colocalized MAP2 expression to the total number of MAP2-positive cells was determined after 14 days of culture for UNT, STS- and TNFα/CHX-treated primary cortical neurons derived from embryos of CRE3 and N casp8 −/− mice. Cells were counted on 15 random fluorescent images (×20) from 3 experiments. ( C–I ) Bar graphs present average percentages of MAP2 ( C ), NeuN ( D ), cleaved caspase 3 ( E ), GFAP ( G ), S100 ( H ), Iba1 ( I ) immunopositive staining and mean percentage of TUNEL positive signaling ( F ) in cortices (left) and basal ganglia (right) of untreated and TRAIL-treated cultured coronal brain slices derived from CRE3, casp8 fl/fl (Casp8 line) and N casp8 −/− mice after 3 days of culture. Tables 1 and 2 provide the results of statistical analyses.

    Article Snippet: TRAIL is known to transmit the apoptotic signals through death receptors that directly activate caspase 8 .

    Techniques: Cell Culture, Staining, Derivative Assay, Mouse Assay, Expressing, TUNEL Assay

    Caspase 8 deficiency protects neurons in in vivo KA-induced seizure mouse model. ( A ) Mouse survival was monitored for 24 hours after exposure of control (n = 8) and N casp8 −/− mice (n = 11) to KA. Non-linear x-axis corresponds to time following KA injection. ( B ) Seizure scores for control and N casp8 −/− mice were determined every 15 min for 2 h and at longer intervals thereafter [40] [41] . Percentage of TUNEL-positive neurons ( C ) and density of degenerating neurons (Masson's trichrome stain) ( D ) were assessed in the hippocampi of the control and N casp8 −/− mice (mean±SEM). P values result from Student's t-test ( B : * p = 0.008; C : * p = 0.002; D : * p = 0.005). Colorimetric intensity measurements were applied to Masson's trichrome-stained control ( E ) and N casp8 −/− ( F ) hippocampi to obtain density of degenerating neurons in the annotated area (mark-up images). Arrows indicate hippocampal CA3–CA4 sectors and the hilus ( E ) and the CA3 sector ( F ). Apoptotic neurons were visualized by TUNEL assay ( G, H ), and cleaved-caspase 3 immunostaining ( I–K ) as presented on virtual ( I, K ) and mark-up ( G, H, J ) images of the hippocampal regions of the control ( G, I, J ) and N casp8 −/− ( H, K ) mice. Brown color (DAB) on digital images and red, orange and yellow pixels on mark-up images denote positive staining. The arrow indicates TUNEL positive neurons in the CA3–CA4 sector.

    Journal: PLoS ONE

    Article Title: Neuronal Deletion of Caspase 8 Protects against Brain Injury in Mouse Models of Controlled Cortical Impact and Kainic Acid-Induced Excitotoxicity

    doi: 10.1371/journal.pone.0024341

    Figure Lengend Snippet: Caspase 8 deficiency protects neurons in in vivo KA-induced seizure mouse model. ( A ) Mouse survival was monitored for 24 hours after exposure of control (n = 8) and N casp8 −/− mice (n = 11) to KA. Non-linear x-axis corresponds to time following KA injection. ( B ) Seizure scores for control and N casp8 −/− mice were determined every 15 min for 2 h and at longer intervals thereafter [40] [41] . Percentage of TUNEL-positive neurons ( C ) and density of degenerating neurons (Masson's trichrome stain) ( D ) were assessed in the hippocampi of the control and N casp8 −/− mice (mean±SEM). P values result from Student's t-test ( B : * p = 0.008; C : * p = 0.002; D : * p = 0.005). Colorimetric intensity measurements were applied to Masson's trichrome-stained control ( E ) and N casp8 −/− ( F ) hippocampi to obtain density of degenerating neurons in the annotated area (mark-up images). Arrows indicate hippocampal CA3–CA4 sectors and the hilus ( E ) and the CA3 sector ( F ). Apoptotic neurons were visualized by TUNEL assay ( G, H ), and cleaved-caspase 3 immunostaining ( I–K ) as presented on virtual ( I, K ) and mark-up ( G, H, J ) images of the hippocampal regions of the control ( G, I, J ) and N casp8 −/− ( H, K ) mice. Brown color (DAB) on digital images and red, orange and yellow pixels on mark-up images denote positive staining. The arrow indicates TUNEL positive neurons in the CA3–CA4 sector.

    Article Snippet: TRAIL is known to transmit the apoptotic signals through death receptors that directly activate caspase 8 .

    Techniques: In Vivo, Mouse Assay, Injection, TUNEL Assay, Staining, Immunostaining

    Generation of mouse line (Ncasp8 −/− ) with neuron-specific ablation of caspase8. ( A ) PCR was performed using 1 µg tail genomic DNA for transgenic CRE3 and caspase 8 flox animals. PCR products were generated using amplification primers specific to either the cre or casp8 genes. PCR products were analyzed by agarose gel-electrophoresis and ethidium bromide staining. Arrows indicate the cre PCR product of 750 bp and casp8 PCR products of 800 bp for floxed genes and 650 bp for wild-type casp8 . ( B ) Brain-specific recombination of floxed casp8 genes in CRE3 mice. Genomic DNA was isolated from various tissues of CRE3 mice containing either wild-type casp8 genes or homozygous floxed casp8 genes (N casp8 −/− ). PCR was performed using primers specific for casp8 (top) or the cre transgene (bottom). Brain tissues included cortex (lanes #1), thalamus/basal ganglia (lanes #2), and brain stem (lanes #3). Arrows indicate the expected PCR products for unrecombined (fl/fl) at 650 bp, recombined caspase8 gene knockout at ∼200 bp (−/−), and wild-type unfloxed (+/+) c asp8 genes at ∼800 bp. ( C ) SDS-PAGE immunoblot analysis of caspase 8 protein levels in mouse brain tissues. Brain samples from cortex (C), cerebellum (CB) or basal ganglia (BG) of casp8 fl/fl -CRE3 mice were compared with CRE3 and casp8 fl/fl animals. Lysates were normalized for total protein content (30 µg/lane) and analyzed by SDS-PAGE/immunoblotting using antibodies specific for mouse caspase 8 (top), or β-actin (bottom), using a multiple antigen detection method [39] . ( D–K ) Examples are provided of immunostaining for mouse caspase 8 in the brains of CRE3 ( D–G ) and N casp8 −/− mice ( H–K ). Antibody detection was accomplished using diaminobenzidine (DAB) chromogen (brown); nuclei were counterstained with hematoxylin (blue). Magnification scale bar = ∼100 µm.

    Journal: PLoS ONE

    Article Title: Neuronal Deletion of Caspase 8 Protects against Brain Injury in Mouse Models of Controlled Cortical Impact and Kainic Acid-Induced Excitotoxicity

    doi: 10.1371/journal.pone.0024341

    Figure Lengend Snippet: Generation of mouse line (Ncasp8 −/− ) with neuron-specific ablation of caspase8. ( A ) PCR was performed using 1 µg tail genomic DNA for transgenic CRE3 and caspase 8 flox animals. PCR products were generated using amplification primers specific to either the cre or casp8 genes. PCR products were analyzed by agarose gel-electrophoresis and ethidium bromide staining. Arrows indicate the cre PCR product of 750 bp and casp8 PCR products of 800 bp for floxed genes and 650 bp for wild-type casp8 . ( B ) Brain-specific recombination of floxed casp8 genes in CRE3 mice. Genomic DNA was isolated from various tissues of CRE3 mice containing either wild-type casp8 genes or homozygous floxed casp8 genes (N casp8 −/− ). PCR was performed using primers specific for casp8 (top) or the cre transgene (bottom). Brain tissues included cortex (lanes #1), thalamus/basal ganglia (lanes #2), and brain stem (lanes #3). Arrows indicate the expected PCR products for unrecombined (fl/fl) at 650 bp, recombined caspase8 gene knockout at ∼200 bp (−/−), and wild-type unfloxed (+/+) c asp8 genes at ∼800 bp. ( C ) SDS-PAGE immunoblot analysis of caspase 8 protein levels in mouse brain tissues. Brain samples from cortex (C), cerebellum (CB) or basal ganglia (BG) of casp8 fl/fl -CRE3 mice were compared with CRE3 and casp8 fl/fl animals. Lysates were normalized for total protein content (30 µg/lane) and analyzed by SDS-PAGE/immunoblotting using antibodies specific for mouse caspase 8 (top), or β-actin (bottom), using a multiple antigen detection method [39] . ( D–K ) Examples are provided of immunostaining for mouse caspase 8 in the brains of CRE3 ( D–G ) and N casp8 −/− mice ( H–K ). Antibody detection was accomplished using diaminobenzidine (DAB) chromogen (brown); nuclei were counterstained with hematoxylin (blue). Magnification scale bar = ∼100 µm.

    Article Snippet: TRAIL is known to transmit the apoptotic signals through death receptors that directly activate caspase 8 .

    Techniques: Polymerase Chain Reaction, Transgenic Assay, Generated, Amplification, Agarose Gel Electrophoresis, Staining, Mouse Assay, Isolation, Gene Knockout, SDS Page, Immunostaining

    Y465 phosphomimetic modification of caspase-8A is necessary but not sufficient for Src activation. A) HEK293 cells were co-transfected with Y465E GFP-caspase-8A mutant and Y527F Src for 24 hours. Whole cell lysates were subjected to Western blot analysis with an anti-phosphotyrosine antibody (non-reactive to glutamic acid residue in phosphomimetic mutant) and an anti-GFP antibody. Y465E of GFP-caspase-8A was tyrosine phosphorylated. B) HEK293 cells were co-transfected with Y465E GFP-caspase-8A mutant and Y527F Src for 24 hours. GFP-caspase-8A IP was sent for LC-MS/MS analysis. C) This is a schematic figure showing the location of tyrosine residues that we mutated. D, E) We transfected HEK293 cells with Y527F Src and various GFP-caspase-8A mutants for 24 hours. Whole cell lysates were subjected to Western blot analysis with pY416Src antibody and Src antibody. N = 4, p = 0.001 (Y465E vs Y397E/Y465F); p = 0.139 (Y465E vs Y397F/Y465E). F, G) We transfected HEK293 cells with Y527F Src and pEGFP empty plasmid or pEGFP fusion plasmid with WT or various mutants of GFP-caspase-8 for 24 hours. We then immunoprecipitated transfected avian Src and resolve the immunoprecipitate on SDS-PAGE for Western blot analysis. N = 3, p = 0.04.

    Journal: PLoS ONE

    Article Title: Tyrosine Phosphorylation of Caspase-8 Abrogates Its Apoptotic Activity and Promotes Activation of c-Src

    doi: 10.1371/journal.pone.0153946

    Figure Lengend Snippet: Y465 phosphomimetic modification of caspase-8A is necessary but not sufficient for Src activation. A) HEK293 cells were co-transfected with Y465E GFP-caspase-8A mutant and Y527F Src for 24 hours. Whole cell lysates were subjected to Western blot analysis with an anti-phosphotyrosine antibody (non-reactive to glutamic acid residue in phosphomimetic mutant) and an anti-GFP antibody. Y465E of GFP-caspase-8A was tyrosine phosphorylated. B) HEK293 cells were co-transfected with Y465E GFP-caspase-8A mutant and Y527F Src for 24 hours. GFP-caspase-8A IP was sent for LC-MS/MS analysis. C) This is a schematic figure showing the location of tyrosine residues that we mutated. D, E) We transfected HEK293 cells with Y527F Src and various GFP-caspase-8A mutants for 24 hours. Whole cell lysates were subjected to Western blot analysis with pY416Src antibody and Src antibody. N = 4, p = 0.001 (Y465E vs Y397E/Y465F); p = 0.139 (Y465E vs Y397F/Y465E). F, G) We transfected HEK293 cells with Y527F Src and pEGFP empty plasmid or pEGFP fusion plasmid with WT or various mutants of GFP-caspase-8 for 24 hours. We then immunoprecipitated transfected avian Src and resolve the immunoprecipitate on SDS-PAGE for Western blot analysis. N = 3, p = 0.04.

    Article Snippet: In the presence of caspase-8 inhibitor, the GFP-caspase-8A tyrosine phosphorylation signal was much stronger , suggesting that Src-dependent tyrosine phosphorylation of caspase-8 was enhanced in the absence of caspase-8 activity.

    Techniques: Modification, Activation Assay, Transfection, Mutagenesis, Western Blot, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Plasmid Preparation, Immunoprecipitation, SDS Page

    Inhibition of caspase-8 activity enhances Src-dependent tyrosine phosphorylation of caspase-8. A) HEK293 cells were transfected with GFP-caspase-8A (WT or C377S inactive mutant) with or without Y527F Src for 24 hours. Whole cell lysates were subjected to Western blot analysis with anti-phospho-tyrosine antibody and anti-GFP antibody. B) HEK293 cells were transfected with GFP-caspase-8A (WT or C377S inactive mutant) with Y527F Src for 24 hours. GFP-caspase-8A was immunoprecipitated with anti-GFP antibody and precipitates were probed with anti-phospho-tyrosine antibody and anti-GFP antibody. C) HEK293 cells were transfected with WT GFP-caspase-8A and Y527F Src for 9 hours followed by treatment with DMSO vehicle control or caspase-8 inhibitor (20 μM) for 15 hours. Whole cell lysates were subjected to Western blot analysis with anti-phospho-tyrosine antibody and anti-GFP antibody.

    Journal: PLoS ONE

    Article Title: Tyrosine Phosphorylation of Caspase-8 Abrogates Its Apoptotic Activity and Promotes Activation of c-Src

    doi: 10.1371/journal.pone.0153946

    Figure Lengend Snippet: Inhibition of caspase-8 activity enhances Src-dependent tyrosine phosphorylation of caspase-8. A) HEK293 cells were transfected with GFP-caspase-8A (WT or C377S inactive mutant) with or without Y527F Src for 24 hours. Whole cell lysates were subjected to Western blot analysis with anti-phospho-tyrosine antibody and anti-GFP antibody. B) HEK293 cells were transfected with GFP-caspase-8A (WT or C377S inactive mutant) with Y527F Src for 24 hours. GFP-caspase-8A was immunoprecipitated with anti-GFP antibody and precipitates were probed with anti-phospho-tyrosine antibody and anti-GFP antibody. C) HEK293 cells were transfected with WT GFP-caspase-8A and Y527F Src for 9 hours followed by treatment with DMSO vehicle control or caspase-8 inhibitor (20 μM) for 15 hours. Whole cell lysates were subjected to Western blot analysis with anti-phospho-tyrosine antibody and anti-GFP antibody.

    Article Snippet: In the presence of caspase-8 inhibitor, the GFP-caspase-8A tyrosine phosphorylation signal was much stronger , suggesting that Src-dependent tyrosine phosphorylation of caspase-8 was enhanced in the absence of caspase-8 activity.

    Techniques: Inhibition, Activity Assay, Transfection, Mutagenesis, Western Blot, Immunoprecipitation

    Y465 phosphomimetic mutation inhibits caspase-8A cleavage. A) Upon activation, caspase-8 is autocatalytically cleaved first after D391, and then after D233. Using an anti-GFP antibody that binds to the N-terminus of the molecule, a 68-kDa fragment is visualized when GFP-caspase-8A is cleaved after D391, whereas a 50-kDa fragment is visualized when GFP-caspase-8A is cleaved after D233. B) Using an anti-caspase-8 antibody that binds to the C-terminus of the molecule, a 30-kDa fragment is visualized when GFP-caspase-8A is cleaved after D233, whereas a 12-kDa fragment is visualized when GFP-caspase-8A is cleaved after D391. A 55-kDa band represents endogenous caspase-8. C) HEK293 cells were transfected with GFP-caspase-8A WT or mutants for 24 hours. Whole cell lysates were subjected to Western blot analysis with anti-GFP antibody that recognizes the N-terminal end of GFP-caspase-8A. D) Whole cell lysates were subjected to Western blot analysis with anti-caspase-8 antibody that recognizes the C-terminal end of GFP-caspase-8A. E) Both the inactive C377S and Y465E GFP-caspase-8A mutants failed to undergo cleavage at D391, the essential step of the activation of caspase-8. F-H) HEK293 cells were transfected with GFP-caspase-8B (F), Myc-caspase-8B (G), GFP-caspase-8A (H) WT or mutants for 24 hours. Whole cell lysates were subjected to Western blot analysis with anti-GFP antibody that recognizes the N-terminal end of GFP-caspase-8 and anti-Myc antibody that recognizes the C-terminal end of GFP-caspase-8 (active caspase-8).

    Journal: PLoS ONE

    Article Title: Tyrosine Phosphorylation of Caspase-8 Abrogates Its Apoptotic Activity and Promotes Activation of c-Src

    doi: 10.1371/journal.pone.0153946

    Figure Lengend Snippet: Y465 phosphomimetic mutation inhibits caspase-8A cleavage. A) Upon activation, caspase-8 is autocatalytically cleaved first after D391, and then after D233. Using an anti-GFP antibody that binds to the N-terminus of the molecule, a 68-kDa fragment is visualized when GFP-caspase-8A is cleaved after D391, whereas a 50-kDa fragment is visualized when GFP-caspase-8A is cleaved after D233. B) Using an anti-caspase-8 antibody that binds to the C-terminus of the molecule, a 30-kDa fragment is visualized when GFP-caspase-8A is cleaved after D233, whereas a 12-kDa fragment is visualized when GFP-caspase-8A is cleaved after D391. A 55-kDa band represents endogenous caspase-8. C) HEK293 cells were transfected with GFP-caspase-8A WT or mutants for 24 hours. Whole cell lysates were subjected to Western blot analysis with anti-GFP antibody that recognizes the N-terminal end of GFP-caspase-8A. D) Whole cell lysates were subjected to Western blot analysis with anti-caspase-8 antibody that recognizes the C-terminal end of GFP-caspase-8A. E) Both the inactive C377S and Y465E GFP-caspase-8A mutants failed to undergo cleavage at D391, the essential step of the activation of caspase-8. F-H) HEK293 cells were transfected with GFP-caspase-8B (F), Myc-caspase-8B (G), GFP-caspase-8A (H) WT or mutants for 24 hours. Whole cell lysates were subjected to Western blot analysis with anti-GFP antibody that recognizes the N-terminal end of GFP-caspase-8 and anti-Myc antibody that recognizes the C-terminal end of GFP-caspase-8 (active caspase-8).

    Article Snippet: In the presence of caspase-8 inhibitor, the GFP-caspase-8A tyrosine phosphorylation signal was much stronger , suggesting that Src-dependent tyrosine phosphorylation of caspase-8 was enhanced in the absence of caspase-8 activity.

    Techniques: Mutagenesis, Activation Assay, Transfection, Western Blot

    Model depicting the etoposide-induced apoptotic signaling pathway in SK-N-AS cells. Etoposide induces the mitochondrial cytochrome c release, leading to the caspase-9-dependent activation of caspase-3. The activation of caspase-3 induces the cleavage of PKCδ, and active PKCδ processes caspase-3 by a positive-feedback mechanism. The activation of caspase-3 leads to the processing of caspase-8, and the expression of caspase-8 and caspase-2 is required for the activation of each other downstream of caspase-3. The etoposide-induced activation of caspase-8 leads to the processing of caspase-6 and apoptosis. Rottlerin inhibits etoposide-induced apoptotic signaling by preventing the PKCδ-mediated activation of caspase-3 and by causing the degradation of caspase-2, which inhibits caspase-8 activation.

    Journal: Molecular Pharmacology

    Article Title: Etoposide Induces Protein Kinase C?- and Caspase-3-Dependent Apoptosis in Neuroblastoma Cancer Cells

    doi: 10.1124/mol.109.054999

    Figure Lengend Snippet: Model depicting the etoposide-induced apoptotic signaling pathway in SK-N-AS cells. Etoposide induces the mitochondrial cytochrome c release, leading to the caspase-9-dependent activation of caspase-3. The activation of caspase-3 induces the cleavage of PKCδ, and active PKCδ processes caspase-3 by a positive-feedback mechanism. The activation of caspase-3 leads to the processing of caspase-8, and the expression of caspase-8 and caspase-2 is required for the activation of each other downstream of caspase-3. The etoposide-induced activation of caspase-8 leads to the processing of caspase-6 and apoptosis. Rottlerin inhibits etoposide-induced apoptotic signaling by preventing the PKCδ-mediated activation of caspase-3 and by causing the degradation of caspase-2, which inhibits caspase-8 activation.

    Article Snippet: In this study, the following primary antibodies were used: anti-caspase-8, anti-caspase-6, anti-PKCδ (BD Biosciences, San Jose, CA), anti-caspase-9, anti-caspase-3 (Cell Signaling Technology, Danvers, MA), anti-caspase-2 (Assay Designs, Inc., Ann Arbor, MI), and anti-β-actin clone AC-74 (Sigma-Aldrich, St. Louis, MO).

    Techniques: Activation Assay, Expressing

    Etoposide induces caspase-8-dependent apoptosis. A, immunoblot analysis of caspase-8 and β-actin in cell lysates treated with 50 μM etoposide with or without 20 μM caspase-3 inhibitor (z-DEVD-fmk) for 48 h. B, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-3-specific siRNA (C3) for 48 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-3, caspase-8, and β-actin levels were determined by immunoblotting. C, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-3, and β-actin levels were determined by immunoblotting. D, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and the percentage of apoptotic cells were quantified by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.

    Journal: Molecular Pharmacology

    Article Title: Etoposide Induces Protein Kinase C?- and Caspase-3-Dependent Apoptosis in Neuroblastoma Cancer Cells

    doi: 10.1124/mol.109.054999

    Figure Lengend Snippet: Etoposide induces caspase-8-dependent apoptosis. A, immunoblot analysis of caspase-8 and β-actin in cell lysates treated with 50 μM etoposide with or without 20 μM caspase-3 inhibitor (z-DEVD-fmk) for 48 h. B, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-3-specific siRNA (C3) for 48 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-3, caspase-8, and β-actin levels were determined by immunoblotting. C, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-3, and β-actin levels were determined by immunoblotting. D, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and the percentage of apoptotic cells were quantified by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.

    Article Snippet: In this study, the following primary antibodies were used: anti-caspase-8, anti-caspase-6, anti-PKCδ (BD Biosciences, San Jose, CA), anti-caspase-9, anti-caspase-3 (Cell Signaling Technology, Danvers, MA), anti-caspase-2 (Assay Designs, Inc., Ann Arbor, MI), and anti-β-actin clone AC-74 (Sigma-Aldrich, St. Louis, MO).

    Techniques: Transfection, Staining

    Etoposide induces the caspase-8-dependent activation of caspase-6 and apoptosis. A, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-6, and β-actin levels were determined by immunoblotting. B, cells were treated with 50 μM etoposide with or without 20 μM caspase-6 inhibitor (z-VEID-fmk) for 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei stained with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.

    Journal: Molecular Pharmacology

    Article Title: Etoposide Induces Protein Kinase C?- and Caspase-3-Dependent Apoptosis in Neuroblastoma Cancer Cells

    doi: 10.1124/mol.109.054999

    Figure Lengend Snippet: Etoposide induces the caspase-8-dependent activation of caspase-6 and apoptosis. A, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-6, and β-actin levels were determined by immunoblotting. B, cells were treated with 50 μM etoposide with or without 20 μM caspase-6 inhibitor (z-VEID-fmk) for 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei stained with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts.

    Article Snippet: In this study, the following primary antibodies were used: anti-caspase-8, anti-caspase-6, anti-PKCδ (BD Biosciences, San Jose, CA), anti-caspase-9, anti-caspase-3 (Cell Signaling Technology, Danvers, MA), anti-caspase-2 (Assay Designs, Inc., Ann Arbor, MI), and anti-β-actin clone AC-74 (Sigma-Aldrich, St. Louis, MO).

    Techniques: Activation Assay, Transfection, Staining

    Etoposide induces caspase-2-dependent apoptosis. A, immunoblot analysis of caspase-2 and β-actin in cell lysates treated with or without 2 μM rottlerin, 50 μM etoposide, or 2 μM rottlerin and 50 μM etoposide for 48 h. B, cells were transfected with nontargeting siRNA (SC) or caspase-2-specific siRNA (C2) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained, and caspase-2, caspase-8, caspase-3, and β-actin levels were determined by immunoblotting. C, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-2-specific siRNA (C2) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts. D, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-2, and β-actin levels were determined by immunoblotting. * , a nonspecific protein that is recognized by the caspase-2-specific antibody.

    Journal: Molecular Pharmacology

    Article Title: Etoposide Induces Protein Kinase C?- and Caspase-3-Dependent Apoptosis in Neuroblastoma Cancer Cells

    doi: 10.1124/mol.109.054999

    Figure Lengend Snippet: Etoposide induces caspase-2-dependent apoptosis. A, immunoblot analysis of caspase-2 and β-actin in cell lysates treated with or without 2 μM rottlerin, 50 μM etoposide, or 2 μM rottlerin and 50 μM etoposide for 48 h. B, cells were transfected with nontargeting siRNA (SC) or caspase-2-specific siRNA (C2) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained, and caspase-2, caspase-8, caspase-3, and β-actin levels were determined by immunoblotting. C, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-2-specific siRNA (C2) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and the percentage of apoptotic cells was quantified by counting fragmented nuclei after staining with Hoechst 33342 among 200 cells. Shown are representative apoptosis rates from three independent counts. D, cells were transfected with 100 nM nontargeting siRNA (SC) or caspase-8-specific siRNA (C8) for 72 h followed by the addition of 50 μM etoposide for an additional 48 h, and cell lysates were obtained and caspase-8, caspase-2, and β-actin levels were determined by immunoblotting. * , a nonspecific protein that is recognized by the caspase-2-specific antibody.

    Article Snippet: In this study, the following primary antibodies were used: anti-caspase-8, anti-caspase-6, anti-PKCδ (BD Biosciences, San Jose, CA), anti-caspase-9, anti-caspase-3 (Cell Signaling Technology, Danvers, MA), anti-caspase-2 (Assay Designs, Inc., Ann Arbor, MI), and anti-β-actin clone AC-74 (Sigma-Aldrich, St. Louis, MO).

    Techniques: Transfection, Staining

    Inactivation of caspase-dependent apoptosis in ischemic cardiomyocytes treated with an AKT2 inhibitor. ( A ) Initiator caspase-8 activity in extracts of cardiomyocytes with con, an AKT2 inhibitor, and ischemic cardiomyocytes treated with or without AKT2 inhibitor and/or zVAD; ( B ) Quantitative real time PCR of cFlip transcript in cardiomyocytes with the same treatment as in A. zVAD: pan-caspase inhibitor z-VAD-fmk, 100 μM. The bars represent the mean ± SEM of three independent experiments. * p

    Journal: International Journal of Molecular Sciences

    Article Title: AKT2 Blocks Nucleus Translocation of Apoptosis-Inducing Factor (AIF) and Endonuclease G (EndoG) While Promoting Caspase Activation during Cardiac Ischemia

    doi: 10.3390/ijms18030565

    Figure Lengend Snippet: Inactivation of caspase-dependent apoptosis in ischemic cardiomyocytes treated with an AKT2 inhibitor. ( A ) Initiator caspase-8 activity in extracts of cardiomyocytes with con, an AKT2 inhibitor, and ischemic cardiomyocytes treated with or without AKT2 inhibitor and/or zVAD; ( B ) Quantitative real time PCR of cFlip transcript in cardiomyocytes with the same treatment as in A. zVAD: pan-caspase inhibitor z-VAD-fmk, 100 μM. The bars represent the mean ± SEM of three independent experiments. * p

    Article Snippet: Enzymatic Caspase Activity Assay Caspases activities were measured using the caspase-8, -9 and -3 activity assay kit (Beyotime, C1115, C1151 and C1157) according to the manufacturer’s instructions.

    Techniques: Activity Assay, Real-time Polymerase Chain Reaction

    Caspase-8 and -9 are processed in F. novicida -infected Casp1 KO macrophages and their inhibition reverts caspase-1-independent cell death. Caspase-8 ( a ) and -9 ( b ) cleavage were investigated by immunoblot analysis in WT, Casp1 KO and ASC KO BMM infected

    Journal: Cell Death and Differentiation

    Article Title: AIM2/ASC triggers caspase-8-dependent apoptosis in Francisella-infected caspase-1-deficient macrophages

    doi: 10.1038/cdd.2012.51

    Figure Lengend Snippet: Caspase-8 and -9 are processed in F. novicida -infected Casp1 KO macrophages and their inhibition reverts caspase-1-independent cell death. Caspase-8 ( a ) and -9 ( b ) cleavage were investigated by immunoblot analysis in WT, Casp1 KO and ASC KO BMM infected

    Article Snippet: Antibodies anti–caspase–2 (kindly provided by Dr. J Yuan, 103 dilution), anti caspase-3 (sc-136219, Santa Cruz Biotechnology, Inc., Heidelberg, Germany, 103 dilution), anti caspase-8 (ALX-804-447, Enzo Life Sciences, Villeurbanne, France, 3 × 103 dilution), anti caspase-9 (M054, MBL, Woburn, MA, USA, 104 dilution), anti IkB- α (9242, Cell Signaling, Beverly, MA, USA, 103 dilution) were used.

    Techniques: Infection, Inhibition

    Caspase-8 functionally interacts with ASC within the AIM2/ASC speck. ( a ) Diagram of inflammasome-like pathway reconstitution in 293T cells. ( b ) The different initiator caspase prodomains (CARD domains for caspase-1, -2, -9, -11, -12 and DED domain for

    Journal: Cell Death and Differentiation

    Article Title: AIM2/ASC triggers caspase-8-dependent apoptosis in Francisella-infected caspase-1-deficient macrophages

    doi: 10.1038/cdd.2012.51

    Figure Lengend Snippet: Caspase-8 functionally interacts with ASC within the AIM2/ASC speck. ( a ) Diagram of inflammasome-like pathway reconstitution in 293T cells. ( b ) The different initiator caspase prodomains (CARD domains for caspase-1, -2, -9, -11, -12 and DED domain for

    Article Snippet: Antibodies anti–caspase–2 (kindly provided by Dr. J Yuan, 103 dilution), anti caspase-3 (sc-136219, Santa Cruz Biotechnology, Inc., Heidelberg, Germany, 103 dilution), anti caspase-8 (ALX-804-447, Enzo Life Sciences, Villeurbanne, France, 3 × 103 dilution), anti caspase-9 (M054, MBL, Woburn, MA, USA, 104 dilution), anti IkB- α (9242, Cell Signaling, Beverly, MA, USA, 103 dilution) were used.

    Techniques: