rabbit mab cleaved caspase 8 asp387  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit mab cleaved caspase 8 asp387
    NLRC4 regulates early IL-1β release after P. brasiliensis infection WT and Nlrc4 −/− and mice were intravenously infected with 1x10 6 of viable Pb18 yeasts. (A) Scatterplots with bars show the fungal load in lung of WT and Nlrc4 −/− mice at 7 dpi. Results represent a pool of three independent experiments. (B) Scatterplots with bars show the quantification of IL-1β at 7 in the lungs of WT and Nlrc4 −/− mice. (C) Western blotting analysis detecting IL-1β cleavage (p17) in lung homogenates from WT and Nlrc4 −/− mice at 7 dpi. The intensities of IL-1β protein were quantified using iBright™ CL1500 Imaging System. (D) IL-1β expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 50 μM – left panel). Each column represents the mean ± SD. (E) Caspase-1 expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 20 μM – right panel). Each column represents the mean ± SD. (F) Active caspase-1 (p20) was detected by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-1 were quantified using iBright™ CL1500 Imaging System. (G) Active <t>caspase-8</t> (p18) was detect by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-8 were quantified using iBright™ CL1500 Imaging System. (H) Stacked bars show the percentage of neutrophils (Ly6G + CD11b + ), monocytes (Ly6G − , Ly6C + CD11b + ), macrophages (CD11b + F4/80 + MHC-II + ), and dendritic cells (CD11c + CD11b + MHC-II + ). (I) Contour plots show representative flow cytometric data and scatterplots with bars show the absolute numbers of macrophages and dendritic cells gated in CD45 + IL-1β + cells at 7 dpi (n = 5 infected mice per group at 7 dpi).Data represent one of two independent experiments ( n = 3– 5 infected mice per group at each time point). Data are expressed as mean ± SD. Statistically significant differences were evaluated with ANOVA followed by Bonferroni's multiple comparisons test or unpaired t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also <xref ref-type=Figure S1 . " width="250" height="auto" />
    Rabbit Mab Cleaved Caspase 8 Asp387, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit mab cleaved caspase 8 asp387/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit mab cleaved caspase 8 asp387 - by Bioz Stars, 2023-02
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    Images

    1) Product Images from "NLRC4 inhibits NLRP3 inflammasome and abrogates effective antifungal CD8 + T cell responses"

    Article Title: NLRC4 inhibits NLRP3 inflammasome and abrogates effective antifungal CD8 + T cell responses

    Journal: iScience

    doi: 10.1016/j.isci.2021.102548

    NLRC4 regulates early IL-1β release after P. brasiliensis infection WT and Nlrc4 −/− and mice were intravenously infected with 1x10 6 of viable Pb18 yeasts. (A) Scatterplots with bars show the fungal load in lung of WT and Nlrc4 −/− mice at 7 dpi. Results represent a pool of three independent experiments. (B) Scatterplots with bars show the quantification of IL-1β at 7 in the lungs of WT and Nlrc4 −/− mice. (C) Western blotting analysis detecting IL-1β cleavage (p17) in lung homogenates from WT and Nlrc4 −/− mice at 7 dpi. The intensities of IL-1β protein were quantified using iBright™ CL1500 Imaging System. (D) IL-1β expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 50 μM – left panel). Each column represents the mean ± SD. (E) Caspase-1 expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 20 μM – right panel). Each column represents the mean ± SD. (F) Active caspase-1 (p20) was detected by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-1 were quantified using iBright™ CL1500 Imaging System. (G) Active caspase-8 (p18) was detect by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-8 were quantified using iBright™ CL1500 Imaging System. (H) Stacked bars show the percentage of neutrophils (Ly6G + CD11b + ), monocytes (Ly6G − , Ly6C + CD11b + ), macrophages (CD11b + F4/80 + MHC-II + ), and dendritic cells (CD11c + CD11b + MHC-II + ). (I) Contour plots show representative flow cytometric data and scatterplots with bars show the absolute numbers of macrophages and dendritic cells gated in CD45 + IL-1β + cells at 7 dpi (n = 5 infected mice per group at 7 dpi).Data represent one of two independent experiments ( n = 3– 5 infected mice per group at each time point). Data are expressed as mean ± SD. Statistically significant differences were evaluated with ANOVA followed by Bonferroni's multiple comparisons test or unpaired t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also <xref ref-type=Figure S1 . " title="... quantified using iBright™ CL1500 Imaging System. (G) Active caspase-8 (p18) was detect by Western blotting in the ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: NLRC4 regulates early IL-1β release after P. brasiliensis infection WT and Nlrc4 −/− and mice were intravenously infected with 1x10 6 of viable Pb18 yeasts. (A) Scatterplots with bars show the fungal load in lung of WT and Nlrc4 −/− mice at 7 dpi. Results represent a pool of three independent experiments. (B) Scatterplots with bars show the quantification of IL-1β at 7 in the lungs of WT and Nlrc4 −/− mice. (C) Western blotting analysis detecting IL-1β cleavage (p17) in lung homogenates from WT and Nlrc4 −/− mice at 7 dpi. The intensities of IL-1β protein were quantified using iBright™ CL1500 Imaging System. (D) IL-1β expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 50 μM – left panel). Each column represents the mean ± SD. (E) Caspase-1 expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 20 μM – right panel). Each column represents the mean ± SD. (F) Active caspase-1 (p20) was detected by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-1 were quantified using iBright™ CL1500 Imaging System. (G) Active caspase-8 (p18) was detect by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-8 were quantified using iBright™ CL1500 Imaging System. (H) Stacked bars show the percentage of neutrophils (Ly6G + CD11b + ), monocytes (Ly6G − , Ly6C + CD11b + ), macrophages (CD11b + F4/80 + MHC-II + ), and dendritic cells (CD11c + CD11b + MHC-II + ). (I) Contour plots show representative flow cytometric data and scatterplots with bars show the absolute numbers of macrophages and dendritic cells gated in CD45 + IL-1β + cells at 7 dpi (n = 5 infected mice per group at 7 dpi).Data represent one of two independent experiments ( n = 3– 5 infected mice per group at each time point). Data are expressed as mean ± SD. Statistically significant differences were evaluated with ANOVA followed by Bonferroni's multiple comparisons test or unpaired t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also Figure S1 .

    Techniques Used: Infection, Western Blot, Imaging, Expressing, Immunohistochemistry


    Figure Legend Snippet:

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Bradford Protein Assay, Knock-Out, Double Knockout, Mouse Assay, Software, Imaging

    rabbit mab cleaved caspase 8 asp387  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit mab cleaved caspase 8 asp387
    NLRC4 regulates early IL-1β release after P. brasiliensis infection WT and Nlrc4 −/− and mice were intravenously infected with 1x10 6 of viable Pb18 yeasts. (A) Scatterplots with bars show the fungal load in lung of WT and Nlrc4 −/− mice at 7 dpi. Results represent a pool of three independent experiments. (B) Scatterplots with bars show the quantification of IL-1β at 7 in the lungs of WT and Nlrc4 −/− mice. (C) Western blotting analysis detecting IL-1β cleavage (p17) in lung homogenates from WT and Nlrc4 −/− mice at 7 dpi. The intensities of IL-1β protein were quantified using iBright™ CL1500 Imaging System. (D) IL-1β expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 50 μM – left panel). Each column represents the mean ± SD. (E) Caspase-1 expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 20 μM – right panel). Each column represents the mean ± SD. (F) Active caspase-1 (p20) was detected by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-1 were quantified using iBright™ CL1500 Imaging System. (G) Active <t>caspase-8</t> (p18) was detect by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-8 were quantified using iBright™ CL1500 Imaging System. (H) Stacked bars show the percentage of neutrophils (Ly6G + CD11b + ), monocytes (Ly6G − , Ly6C + CD11b + ), macrophages (CD11b + F4/80 + MHC-II + ), and dendritic cells (CD11c + CD11b + MHC-II + ). (I) Contour plots show representative flow cytometric data and scatterplots with bars show the absolute numbers of macrophages and dendritic cells gated in CD45 + IL-1β + cells at 7 dpi (n = 5 infected mice per group at 7 dpi).Data represent one of two independent experiments ( n = 3– 5 infected mice per group at each time point). Data are expressed as mean ± SD. Statistically significant differences were evaluated with ANOVA followed by Bonferroni's multiple comparisons test or unpaired t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also <xref ref-type=Figure S1 . " width="250" height="auto" />
    Rabbit Mab Cleaved Caspase 8 Asp387, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit mab cleaved caspase 8 asp387/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit mab cleaved caspase 8 asp387 - by Bioz Stars, 2023-02
    93/100 stars

    Images

    1) Product Images from "NLRC4 inhibits NLRP3 inflammasome and abrogates effective antifungal CD8 + T cell responses"

    Article Title: NLRC4 inhibits NLRP3 inflammasome and abrogates effective antifungal CD8 + T cell responses

    Journal: iScience

    doi: 10.1016/j.isci.2021.102548

    NLRC4 regulates early IL-1β release after P. brasiliensis infection WT and Nlrc4 −/− and mice were intravenously infected with 1x10 6 of viable Pb18 yeasts. (A) Scatterplots with bars show the fungal load in lung of WT and Nlrc4 −/− mice at 7 dpi. Results represent a pool of three independent experiments. (B) Scatterplots with bars show the quantification of IL-1β at 7 in the lungs of WT and Nlrc4 −/− mice. (C) Western blotting analysis detecting IL-1β cleavage (p17) in lung homogenates from WT and Nlrc4 −/− mice at 7 dpi. The intensities of IL-1β protein were quantified using iBright™ CL1500 Imaging System. (D) IL-1β expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 50 μM – left panel). Each column represents the mean ± SD. (E) Caspase-1 expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 20 μM – right panel). Each column represents the mean ± SD. (F) Active caspase-1 (p20) was detected by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-1 were quantified using iBright™ CL1500 Imaging System. (G) Active caspase-8 (p18) was detect by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-8 were quantified using iBright™ CL1500 Imaging System. (H) Stacked bars show the percentage of neutrophils (Ly6G + CD11b + ), monocytes (Ly6G − , Ly6C + CD11b + ), macrophages (CD11b + F4/80 + MHC-II + ), and dendritic cells (CD11c + CD11b + MHC-II + ). (I) Contour plots show representative flow cytometric data and scatterplots with bars show the absolute numbers of macrophages and dendritic cells gated in CD45 + IL-1β + cells at 7 dpi (n = 5 infected mice per group at 7 dpi).Data represent one of two independent experiments ( n = 3– 5 infected mice per group at each time point). Data are expressed as mean ± SD. Statistically significant differences were evaluated with ANOVA followed by Bonferroni's multiple comparisons test or unpaired t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also <xref ref-type=Figure S1 . " title="... quantified using iBright™ CL1500 Imaging System. (G) Active caspase-8 (p18) was detect by Western blotting in the ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: NLRC4 regulates early IL-1β release after P. brasiliensis infection WT and Nlrc4 −/− and mice were intravenously infected with 1x10 6 of viable Pb18 yeasts. (A) Scatterplots with bars show the fungal load in lung of WT and Nlrc4 −/− mice at 7 dpi. Results represent a pool of three independent experiments. (B) Scatterplots with bars show the quantification of IL-1β at 7 in the lungs of WT and Nlrc4 −/− mice. (C) Western blotting analysis detecting IL-1β cleavage (p17) in lung homogenates from WT and Nlrc4 −/− mice at 7 dpi. The intensities of IL-1β protein were quantified using iBright™ CL1500 Imaging System. (D) IL-1β expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 50 μM – left panel). Each column represents the mean ± SD. (E) Caspase-1 expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 20 μM – right panel). Each column represents the mean ± SD. (F) Active caspase-1 (p20) was detected by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-1 were quantified using iBright™ CL1500 Imaging System. (G) Active caspase-8 (p18) was detect by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-8 were quantified using iBright™ CL1500 Imaging System. (H) Stacked bars show the percentage of neutrophils (Ly6G + CD11b + ), monocytes (Ly6G − , Ly6C + CD11b + ), macrophages (CD11b + F4/80 + MHC-II + ), and dendritic cells (CD11c + CD11b + MHC-II + ). (I) Contour plots show representative flow cytometric data and scatterplots with bars show the absolute numbers of macrophages and dendritic cells gated in CD45 + IL-1β + cells at 7 dpi (n = 5 infected mice per group at 7 dpi).Data represent one of two independent experiments ( n = 3– 5 infected mice per group at each time point). Data are expressed as mean ± SD. Statistically significant differences were evaluated with ANOVA followed by Bonferroni's multiple comparisons test or unpaired t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also Figure S1 .

    Techniques Used: Infection, Western Blot, Imaging, Expressing, Immunohistochemistry


    Figure Legend Snippet:

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Bradford Protein Assay, Knock-Out, Double Knockout, Mouse Assay, Software, Imaging

    cleaved caspase 8 asp387 d5b2 xp rabbit mab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cleaved caspase 8 asp387 d5b2 xp rabbit mab
    Antibodies
    Cleaved Caspase 8 Asp387 D5b2 Xp Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cleaved caspase 8 asp387 d5b2 xp rabbit mab/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
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    cleaved caspase 8 asp387 d5b2 xp rabbit mab - by Bioz Stars, 2023-02
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    Images

    1) Product Images from "TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection"

    Article Title: TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection

    Journal: Cellular and Molecular Neurobiology

    doi: 10.1007/s10571-021-01063-w

    Antibodies
    Figure Legend Snippet: Antibodies

    Techniques Used:

    The PPI network for the core enriched genes related to the apoptosis and necroptosis pathways. a , b . PPI network analysis for the core enriched genes of GSEA (as shown in Fig. c, d) showed that caspase-8 and TNF-α were the hub genes for the apoptosis and necroptosis pathways in mouse brains infected by AC, respectively. AC Angiostrongylus cantonensis
    Figure Legend Snippet: The PPI network for the core enriched genes related to the apoptosis and necroptosis pathways. a , b . PPI network analysis for the core enriched genes of GSEA (as shown in Fig. c, d) showed that caspase-8 and TNF-α were the hub genes for the apoptosis and necroptosis pathways in mouse brains infected by AC, respectively. AC Angiostrongylus cantonensis

    Techniques Used: Infection

    TNF-α triggers RIP1/FADD/caspase-8-mediated apoptosis of astrocytes in mouse brains with AC infection. a Relative mRNA level of TNF-α in mouse brains upon AC infection was significantly higher than that in the control group. b TNF-α protein expression level in the mouse brain post infection was remarkably elevated, as revealed by immunofluorescence (left) and relative mean fluorescence intensity (MFI, right). c The lysates of brain tissues from normal control mice or mice post infection were subjected to western blot to determine the protein levels of genes related to the apoptosis signalling pathway (TNF-α, RIP1, caspase-8 and cleaved caspase-8). d – g The protein expression levels in c were quantified via density analysis. h Mice were infected by AC for 21 days and then the lysates of mouse brain tissues were immunoprecipitated with an anti-RIP1 antibody or an IgG antibody and the precipitated complexes were separately analysed by immunoblotting with antibodies against RIP1, RIP3, FADD, caspase-8 and GAPDH. i Cleaved caspase-3 and GFAP (specific marker of astrocytes) were co-localized, as shown by immunofluorescence. j Cleaved caspase-3 and NeuN (specific marker of neurons) showed no co-localization, as shown by immunofluorescence. * p < 0.05, ** p < 0.01, *** p < 0.001 (student’s t test). AC Angiostrongylus cantonensis , Ctrl normal control, INF infected by Angiostrongylus cantonensis
    Figure Legend Snippet: TNF-α triggers RIP1/FADD/caspase-8-mediated apoptosis of astrocytes in mouse brains with AC infection. a Relative mRNA level of TNF-α in mouse brains upon AC infection was significantly higher than that in the control group. b TNF-α protein expression level in the mouse brain post infection was remarkably elevated, as revealed by immunofluorescence (left) and relative mean fluorescence intensity (MFI, right). c The lysates of brain tissues from normal control mice or mice post infection were subjected to western blot to determine the protein levels of genes related to the apoptosis signalling pathway (TNF-α, RIP1, caspase-8 and cleaved caspase-8). d – g The protein expression levels in c were quantified via density analysis. h Mice were infected by AC for 21 days and then the lysates of mouse brain tissues were immunoprecipitated with an anti-RIP1 antibody or an IgG antibody and the precipitated complexes were separately analysed by immunoblotting with antibodies against RIP1, RIP3, FADD, caspase-8 and GAPDH. i Cleaved caspase-3 and GFAP (specific marker of astrocytes) were co-localized, as shown by immunofluorescence. j Cleaved caspase-3 and NeuN (specific marker of neurons) showed no co-localization, as shown by immunofluorescence. * p < 0.05, ** p < 0.01, *** p < 0.001 (student’s t test). AC Angiostrongylus cantonensis , Ctrl normal control, INF infected by Angiostrongylus cantonensis

    Techniques Used: Infection, Expressing, Immunofluorescence, Fluorescence, Western Blot, Immunoprecipitation, Marker

    cleaved caspase 8 asp387 d5b2 rabbit mab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cleaved caspase 8 asp387 d5b2 rabbit mab
    Cleaved Caspase 8 Asp387 D5b2 Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cleaved caspase 8 asp387 d5b2 rabbit mab/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cleaved caspase 8 asp387 d5b2 rabbit mab - by Bioz Stars, 2023-02
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    cleaved caspase 8 asp387  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cleaved caspase 8 asp387
    Reduced DNA Damage And Genetic Instability in Mcl-1 Δhep /TNFR1 −/− and <t>TAK1/Casp8</t> Δhep Mice and Intercrossings (A) Staining for γH2AX (black) and cleaved Casp3 (red), double-positive hepatocytes (black/red arrows). Scale bar, 50 μm. (B) IF staining for γH2AX and Ki67 in wild-type, Mcl-1 Δhep , and Mcl-1 Δhep /TNFR1 −/− mice, as well as TAK1 Δhep , TAK1/Casp8 Δhep , and TAK1 Δhep /RIPK3 −/− mice. Arrowheads indicate cells with positive IF staining. Scale bar, 10 μm. (C) Quantification of Ki67 + and Ki67 + /γH2AX + hepatocytes (n = 4 mice per group, n = 5 for Mcl-1 Δhep mice). (D) Rate of AI in wild-type, Mcl-1 Δhep , and Mcl-1 Δhep /TNFR1 −/− mice, TAK1 Δhep , TAK1 Δhep /RIPK3 −/− , and TAK1/Casp8 Δhep mice (TaqMan copy number assay, each square represents one area of microdissected liver tissue, lines indicate different areas of the same liver; red, AI; black, no AI). Mcl-1 Δhep mice and intercrossings at 2 months; TAK1 Δhep mice and intercrossings at 6 weeks of age. In (C), data are presented as mean ± SEM. Statistical significance was calculated using ANOVA with Bonferroni correction (C), or Fisher's exact test (D). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also <xref ref-type=Figure S5 . " width="250" height="auto" />
    Cleaved Caspase 8 Asp387, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cleaved caspase 8 asp387/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cleaved caspase 8 asp387 - by Bioz Stars, 2023-02
    93/100 stars

    Images

    1) Product Images from "A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development"

    Article Title: A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development

    Journal: Cancer Cell

    doi: 10.1016/j.ccell.2017.08.010

    Reduced DNA Damage And Genetic Instability in Mcl-1 Δhep /TNFR1 −/− and TAK1/Casp8 Δhep Mice and Intercrossings (A) Staining for γH2AX (black) and cleaved Casp3 (red), double-positive hepatocytes (black/red arrows). Scale bar, 50 μm. (B) IF staining for γH2AX and Ki67 in wild-type, Mcl-1 Δhep , and Mcl-1 Δhep /TNFR1 −/− mice, as well as TAK1 Δhep , TAK1/Casp8 Δhep , and TAK1 Δhep /RIPK3 −/− mice. Arrowheads indicate cells with positive IF staining. Scale bar, 10 μm. (C) Quantification of Ki67 + and Ki67 + /γH2AX + hepatocytes (n = 4 mice per group, n = 5 for Mcl-1 Δhep mice). (D) Rate of AI in wild-type, Mcl-1 Δhep , and Mcl-1 Δhep /TNFR1 −/− mice, TAK1 Δhep , TAK1 Δhep /RIPK3 −/− , and TAK1/Casp8 Δhep mice (TaqMan copy number assay, each square represents one area of microdissected liver tissue, lines indicate different areas of the same liver; red, AI; black, no AI). Mcl-1 Δhep mice and intercrossings at 2 months; TAK1 Δhep mice and intercrossings at 6 weeks of age. In (C), data are presented as mean ± SEM. Statistical significance was calculated using ANOVA with Bonferroni correction (C), or Fisher's exact test (D). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also <xref ref-type=Figure S5 . " title="... Genetic Instability in Mcl-1 Δhep /TNFR1 −/− and TAK1/Casp8 Δhep Mice and Intercrossings (A) Staining for γH2AX ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Reduced DNA Damage And Genetic Instability in Mcl-1 Δhep /TNFR1 −/− and TAK1/Casp8 Δhep Mice and Intercrossings (A) Staining for γH2AX (black) and cleaved Casp3 (red), double-positive hepatocytes (black/red arrows). Scale bar, 50 μm. (B) IF staining for γH2AX and Ki67 in wild-type, Mcl-1 Δhep , and Mcl-1 Δhep /TNFR1 −/− mice, as well as TAK1 Δhep , TAK1/Casp8 Δhep , and TAK1 Δhep /RIPK3 −/− mice. Arrowheads indicate cells with positive IF staining. Scale bar, 10 μm. (C) Quantification of Ki67 + and Ki67 + /γH2AX + hepatocytes (n = 4 mice per group, n = 5 for Mcl-1 Δhep mice). (D) Rate of AI in wild-type, Mcl-1 Δhep , and Mcl-1 Δhep /TNFR1 −/− mice, TAK1 Δhep , TAK1 Δhep /RIPK3 −/− , and TAK1/Casp8 Δhep mice (TaqMan copy number assay, each square represents one area of microdissected liver tissue, lines indicate different areas of the same liver; red, AI; black, no AI). Mcl-1 Δhep mice and intercrossings at 2 months; TAK1 Δhep mice and intercrossings at 6 weeks of age. In (C), data are presented as mean ± SEM. Statistical significance was calculated using ANOVA with Bonferroni correction (C), or Fisher's exact test (D). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also Figure S5 .

    Techniques Used: Staining, TaqMan Copy Number Assay

    Detection of Proliferation-Associated DNA Damage after PHX Is Impaired in Casp8 Δhep Mice (A–C) Western blot analysis of whole-liver lysates (A), immunostainings (B), and quantification of γH2AX + hepatocytes 0, 6, 24, and 48 hr post-PHX (C). Scale bar, 50 μm. (D and E) BrdU incorporation combined with γH2AX staining (n = 4). Scale bar, 10 μm. (F and G) PFGE with densitometric quantification to visualize DNA DSB in livers of wild-type mice after PHX (n = 3). (H and I) IF staining (H) and quantification of Ki67 + /γH2AX + hepatocytes in wild-type, TNFR1/2 −/− , RIPK3 −/− , and Casp8 Δhep mice (I). Arrowheads indicate cells with positive IF staining. Scale bar, 10 μm. (J and K) PFGE with densitometric quantification to visualize DNA DSB in livers of Casp8 Δhep mice after PHX. In (C), bar represents mean. In (E), (G), (I), and (K) data are presented as mean ± SEM. In (G), bar indicates the mean. Statistical significance was calculated using ANOVA with Bonferroni correction (C and I) or Student's t test (E, G, and K). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s., not significant. Irrelevant bands were omitted from gels (F and J). Areas in which lanes were omitted are indicated by white space between lanes. See also <xref ref-type=Figure S6 . " title="... Proliferation-Associated DNA Damage after PHX Is Impaired in Casp8 Δhep Mice (A–C) Western blot analysis of whole-liver ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Detection of Proliferation-Associated DNA Damage after PHX Is Impaired in Casp8 Δhep Mice (A–C) Western blot analysis of whole-liver lysates (A), immunostainings (B), and quantification of γH2AX + hepatocytes 0, 6, 24, and 48 hr post-PHX (C). Scale bar, 50 μm. (D and E) BrdU incorporation combined with γH2AX staining (n = 4). Scale bar, 10 μm. (F and G) PFGE with densitometric quantification to visualize DNA DSB in livers of wild-type mice after PHX (n = 3). (H and I) IF staining (H) and quantification of Ki67 + /γH2AX + hepatocytes in wild-type, TNFR1/2 −/− , RIPK3 −/− , and Casp8 Δhep mice (I). Arrowheads indicate cells with positive IF staining. Scale bar, 10 μm. (J and K) PFGE with densitometric quantification to visualize DNA DSB in livers of Casp8 Δhep mice after PHX. In (C), bar represents mean. In (E), (G), (I), and (K) data are presented as mean ± SEM. In (G), bar indicates the mean. Statistical significance was calculated using ANOVA with Bonferroni correction (C and I) or Student's t test (E, G, and K). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s., not significant. Irrelevant bands were omitted from gels (F and J). Areas in which lanes were omitted are indicated by white space between lanes. See also Figure S6 .

    Techniques Used: Western Blot, BrdU Incorporation Assay, Staining

    Caspase-8, RIPK1, FADD, and c-FLIP Are Crucial for Phosphorylation of H2AX in Hepatocytes upon Doxorubicin Treatment (A) IF for γH2AX in untreated wild-type mice and wild-type, Casp8 Δhep , and QVD-OPH-treated wild-type mice following doxorubicin treatment. Arrow heads illustrate γH2AX + foci in nuclei. Scale bar, 10 μm. (B) PFGE on livers of doxorubicin-treated mice. (C) γH2AX staining of doxorubicin-treated wild-type and caspase-8 D387-mutant mice. Scale bar, 50 μm. (D) γH2AX IF staining 12 hr post-doxorubicin-induced DNA damage in hepatocytes of Casp8 −/− /RIPK3 −/− mice (n = 5), RIPK3 −/− mice (n = 4), RIPK1 KD mice (n = 9), RIPK1 −/− RIPK3 −/− FADD −/− (labeled as R1 −/− R3 −/− FADD −/− , n = 2), RIPK1 +/− RIPK3 −/− FADD −/− (labeled as R1 +/− R3 −/− FADD −/− , n = 2), c-FLIP Δhep (n = 6), and TNFR1/2 −/− mice (n = 6). Arrowheads illustrate γH2AX + foci in nuclei. Scale bar, 10 μm. (E) Quantification of IF stainings (A and D). (F) Immunoprecipitation with anti-caspase-8 antibody (upper panel) and immunoblotting of lysates (lower panel), 0–24 hr after doxorubicin (5 μM) treatment. Red box: RIPK1, FADD, and caspase-8 interaction at 1 hr; blue boxes: low-level activation of apoptosis starting at 4 hr post-treatment. (The signal visible in the t = 0 column, cl.PARP lane, does not originate from cl.PARP, but from a lower unspecific band.) Control cells treated for 1 hr with CD95L/FasL (B, beads; L, lysates). (G) Immunoblotting of lysates, 0–24 hr after doxorubicin (5 μM) treatment looking at levels of total and cl.PARP, blue boxes (F and G): low-level activation of apoptosis starting at 4 hr post-treatment. (H) Levels of LUBAC (HOIP, HOIL-1, and SHARPIN), cIAP1, cIAP2, and XIAP in U2OS cells at 15 min (red box) post-doxorubicin stimulation (5 μM). (I) Subcellular fractionation of U2OS cells. (J) RIPK1 and γH2AX IF staining in U2OS cells after doxorubicin treatment. The arrowhead indicates colocalizing signals. Scale bar, 10 μm. Statistical significance was calculated using ANOVA with Bonferroni correction (E). ∗∗∗ p < 0.001. Irrelevant bands were omitted from gels (B). Areas in which lanes were omitted are indicated by white space between lanes. See also <xref ref-type=Figure S7 . " title="Caspase-8, RIPK1, FADD, and c-FLIP Are Crucial for Phosphorylation ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: Caspase-8, RIPK1, FADD, and c-FLIP Are Crucial for Phosphorylation of H2AX in Hepatocytes upon Doxorubicin Treatment (A) IF for γH2AX in untreated wild-type mice and wild-type, Casp8 Δhep , and QVD-OPH-treated wild-type mice following doxorubicin treatment. Arrow heads illustrate γH2AX + foci in nuclei. Scale bar, 10 μm. (B) PFGE on livers of doxorubicin-treated mice. (C) γH2AX staining of doxorubicin-treated wild-type and caspase-8 D387-mutant mice. Scale bar, 50 μm. (D) γH2AX IF staining 12 hr post-doxorubicin-induced DNA damage in hepatocytes of Casp8 −/− /RIPK3 −/− mice (n = 5), RIPK3 −/− mice (n = 4), RIPK1 KD mice (n = 9), RIPK1 −/− RIPK3 −/− FADD −/− (labeled as R1 −/− R3 −/− FADD −/− , n = 2), RIPK1 +/− RIPK3 −/− FADD −/− (labeled as R1 +/− R3 −/− FADD −/− , n = 2), c-FLIP Δhep (n = 6), and TNFR1/2 −/− mice (n = 6). Arrowheads illustrate γH2AX + foci in nuclei. Scale bar, 10 μm. (E) Quantification of IF stainings (A and D). (F) Immunoprecipitation with anti-caspase-8 antibody (upper panel) and immunoblotting of lysates (lower panel), 0–24 hr after doxorubicin (5 μM) treatment. Red box: RIPK1, FADD, and caspase-8 interaction at 1 hr; blue boxes: low-level activation of apoptosis starting at 4 hr post-treatment. (The signal visible in the t = 0 column, cl.PARP lane, does not originate from cl.PARP, but from a lower unspecific band.) Control cells treated for 1 hr with CD95L/FasL (B, beads; L, lysates). (G) Immunoblotting of lysates, 0–24 hr after doxorubicin (5 μM) treatment looking at levels of total and cl.PARP, blue boxes (F and G): low-level activation of apoptosis starting at 4 hr post-treatment. (H) Levels of LUBAC (HOIP, HOIL-1, and SHARPIN), cIAP1, cIAP2, and XIAP in U2OS cells at 15 min (red box) post-doxorubicin stimulation (5 μM). (I) Subcellular fractionation of U2OS cells. (J) RIPK1 and γH2AX IF staining in U2OS cells after doxorubicin treatment. The arrowhead indicates colocalizing signals. Scale bar, 10 μm. Statistical significance was calculated using ANOVA with Bonferroni correction (E). ∗∗∗ p < 0.001. Irrelevant bands were omitted from gels (B). Areas in which lanes were omitted are indicated by white space between lanes. See also Figure S7 .

    Techniques Used: Staining, Mutagenesis, Labeling, Immunoprecipitation, Western Blot, Activation Assay, Fractionation

    JNK Is a Downstream Mediator of Caspase-8-, c-FLIP-, and RIPK1-Dependent Phosphorylation of H2AX In Vivo and In Vitro (A) Immunohistochemistry for pCHK1, pCHK2, and pcJUN in livers after doxorubicin treatment. Arrowheads indicate pcJUN-positive nuclei. Scale bar, 50 μm. (B) γH2AX and pJNK co-stainings of livers 12 hr post-doxorubicin treatment. Merged: overlay of DAPI, γH2AX, and pJNK staining. Arrowheads indicate IF signals for γH2AX (green), pJNK (red), or overlapping signals of both (yellow). Scale bar, 10 μm. (C and D) IF stainings (C) and quantification for γH2AX in wild-type and JNK1/2-deficient hepatocytes 12 hr post-doxorubicin treatment (D). Arrowheads indicate IF signals for γH2AX. Scale bar, 10 μm. (E) Analysis of DDR signaling by western blotting of lysates from doxorubicin-treated caspase-8 knockdown cells, JNK inhibitor (SP600125) and ATM inhibitor (KU-55933) pre-treated control cells (U2OS). Red boxes: differences in γH2AX and pJNK activation post-doxorubicin treatment between control cells and lentiviral caspase-8 knockdown and JNK inhibitor treated cells. Statistical analysis was corrected for three tests using the Bonferroni method. See also <xref ref-type=Figure S8 . " title="JNK Is a Downstream Mediator of Caspase-8-, c-FLIP-, and RIPK1-Dependent Phosphorylation of H2AX In Vivo ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: JNK Is a Downstream Mediator of Caspase-8-, c-FLIP-, and RIPK1-Dependent Phosphorylation of H2AX In Vivo and In Vitro (A) Immunohistochemistry for pCHK1, pCHK2, and pcJUN in livers after doxorubicin treatment. Arrowheads indicate pcJUN-positive nuclei. Scale bar, 50 μm. (B) γH2AX and pJNK co-stainings of livers 12 hr post-doxorubicin treatment. Merged: overlay of DAPI, γH2AX, and pJNK staining. Arrowheads indicate IF signals for γH2AX (green), pJNK (red), or overlapping signals of both (yellow). Scale bar, 10 μm. (C and D) IF stainings (C) and quantification for γH2AX in wild-type and JNK1/2-deficient hepatocytes 12 hr post-doxorubicin treatment (D). Arrowheads indicate IF signals for γH2AX. Scale bar, 10 μm. (E) Analysis of DDR signaling by western blotting of lysates from doxorubicin-treated caspase-8 knockdown cells, JNK inhibitor (SP600125) and ATM inhibitor (KU-55933) pre-treated control cells (U2OS). Red boxes: differences in γH2AX and pJNK activation post-doxorubicin treatment between control cells and lentiviral caspase-8 knockdown and JNK inhibitor treated cells. Statistical analysis was corrected for three tests using the Bonferroni method. See also Figure S8 .

    Techniques Used: In Vivo, In Vitro, Immunohistochemistry, Staining, Western Blot, Activation Assay

    Evidence for JNK-Dependent DDR in Human Regenerating Livers and Caspase-8 in Human HCC (A) γH2AX and pJNK co-stainings demonstrating JNK-dependent phosphorylation of H2AX in liver tissue of CLD patients or the left lobe of patients after (right) portal vein ligation and liver transection (PVL/LT). Arrowheads indicate IF signals for γH2AX (green), pJNK (red), or overlapping signals of both (yellow). Scale bar, 10 μm. (B) Overall survival of HCC patients depending on HCC caspase-8 expression level (<mean+1SD; n = 307 patients; >mean+1SD; n = 51 patients, log rank test, statistical analysis was corrected for three tests using the Bonferroni method. The Cancer Genome Atlas [TCGA] cohort). (C) Overall survival of HCC patients depending on HCC caspase-8 methylation status (n = 358 patients, TCGA cohort, log rank test).
    Figure Legend Snippet: Evidence for JNK-Dependent DDR in Human Regenerating Livers and Caspase-8 in Human HCC (A) γH2AX and pJNK co-stainings demonstrating JNK-dependent phosphorylation of H2AX in liver tissue of CLD patients or the left lobe of patients after (right) portal vein ligation and liver transection (PVL/LT). Arrowheads indicate IF signals for γH2AX (green), pJNK (red), or overlapping signals of both (yellow). Scale bar, 10 μm. (B) Overall survival of HCC patients depending on HCC caspase-8 expression level (mean+1SD; n = 51 patients, log rank test, statistical analysis was corrected for three tests using the Bonferroni method. The Cancer Genome Atlas [TCGA] cohort). (C) Overall survival of HCC patients depending on HCC caspase-8 methylation status (n = 358 patients, TCGA cohort, log rank test).

    Techniques Used: Ligation, Expressing, Methylation


    Figure Legend Snippet:

    Techniques Used: Purification, Imaging, Mutagenesis, Recombinant, Staining, SYBR Green Assay, Microarray, RNA Expression, RNA Sequencing Assay, Methylation, Labeling, Software, Light Microscopy

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    Cell Signaling Technology Inc anti caspase 8
    ( A ) Cytosolic and mitochondrial RIPK3 and pro3 <t>caspase-8</t> following H37Rv infection. Equal numbers of human Mφ were infected with H37Rv (MOI 10) and lysed for isolation of cytosolic and mitochondrial fractions after 24 h of infection. Equal amounts of protein were immunoprecipitated (IP) with anti-caspase-8 ab and the levels of RIPK3, RIPK1, and pro-caspase 8 were measured by Western blot analysis. ( B-C ) Time7 dependent translocation of RIPK3 and pro-caspase 8 to the mitochondria. Equal numbers of Mφ were infected with virulent H37Rv or ( B ) avirulent H37Ra ( C ) at MOI 10. The levels of pro9 caspase 8 and RIPK3 were determined by subjecting equal amounts of mitochondria isolated from infected Mφ to Western blotting. VDAC was used as a loading control. ( D ) Colocalization of caspase 8 and RIPK3 with the mitochondria of H37Rv-infected Mφ. Left panels: Colocalization of RIPK3 and pro-caspase 8 with mitochondria 24h after infection visualized by confocal fluorescence microscopy. Human Mφ remained either uninfected or were infected with mCherry- H37Rv (MOI 10) for 24h, fixed and stained with mitotracker (green) and anti-RIPK3 or anti caspase 8 ab (red). Scale bar, 30 μm. On the right side of every panel is the quantification of caspase 8 (top) and of RIPK3 (bottom) associated with the mitochondria. Data were analyzed using nonparametric Student t test ( E ) Phase-contrast images of representative infected and uninfected human primary Mφ after 24 h of infection show approximate location of the plasma membrane and the nucleus as indicated by black and white arrows, respectively. Due to incipient necrosis the plasma membrane of the H37Rv-infected Mφ is not clearly visible. ( F ) BMD-Mφ from WT and RIPK3 −/− mice were infected with H37Rv at an MOI of ~10. After 12 h, expression of cleaved caspase 8 was assessed using flowcytometry. Results are represented as mean ± SD. Data were analyzed using one-way ANOVA. *,**,*** Values of P < 0.05, P <0.01 and P<0.001 , respectively were considered to be significant. Data are representative of 2–3 independent experiments.
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    Images

    1) Product Images from "Bcl-x L mediates RIPK3-dependent necrosis in M. tuberculosis -infected macrophages"

    Article Title: Bcl-x L mediates RIPK3-dependent necrosis in M. tuberculosis -infected macrophages

    Journal: Mucosal immunology

    doi: 10.1038/mi.2017.12

    ( A ) Cytosolic and mitochondrial RIPK3 and pro3 caspase-8 following H37Rv infection. Equal numbers of human Mφ were infected with H37Rv (MOI 10) and lysed for isolation of cytosolic and mitochondrial fractions after 24 h of infection. Equal amounts of protein were immunoprecipitated (IP) with anti-caspase-8 ab and the levels of RIPK3, RIPK1, and pro-caspase 8 were measured by Western blot analysis. ( B-C ) Time7 dependent translocation of RIPK3 and pro-caspase 8 to the mitochondria. Equal numbers of Mφ were infected with virulent H37Rv or ( B ) avirulent H37Ra ( C ) at MOI 10. The levels of pro9 caspase 8 and RIPK3 were determined by subjecting equal amounts of mitochondria isolated from infected Mφ to Western blotting. VDAC was used as a loading control. ( D ) Colocalization of caspase 8 and RIPK3 with the mitochondria of H37Rv-infected Mφ. Left panels: Colocalization of RIPK3 and pro-caspase 8 with mitochondria 24h after infection visualized by confocal fluorescence microscopy. Human Mφ remained either uninfected or were infected with mCherry- H37Rv (MOI 10) for 24h, fixed and stained with mitotracker (green) and anti-RIPK3 or anti caspase 8 ab (red). Scale bar, 30 μm. On the right side of every panel is the quantification of caspase 8 (top) and of RIPK3 (bottom) associated with the mitochondria. Data were analyzed using nonparametric Student t test ( E ) Phase-contrast images of representative infected and uninfected human primary Mφ after 24 h of infection show approximate location of the plasma membrane and the nucleus as indicated by black and white arrows, respectively. Due to incipient necrosis the plasma membrane of the H37Rv-infected Mφ is not clearly visible. ( F ) BMD-Mφ from WT and RIPK3 −/− mice were infected with H37Rv at an MOI of ~10. After 12 h, expression of cleaved caspase 8 was assessed using flowcytometry. Results are represented as mean ± SD. Data were analyzed using one-way ANOVA. *,**,*** Values of P < 0.05, P <0.01 and P<0.001 , respectively were considered to be significant. Data are representative of 2–3 independent experiments.
    Figure Legend Snippet: ( A ) Cytosolic and mitochondrial RIPK3 and pro3 caspase-8 following H37Rv infection. Equal numbers of human Mφ were infected with H37Rv (MOI 10) and lysed for isolation of cytosolic and mitochondrial fractions after 24 h of infection. Equal amounts of protein were immunoprecipitated (IP) with anti-caspase-8 ab and the levels of RIPK3, RIPK1, and pro-caspase 8 were measured by Western blot analysis. ( B-C ) Time7 dependent translocation of RIPK3 and pro-caspase 8 to the mitochondria. Equal numbers of Mφ were infected with virulent H37Rv or ( B ) avirulent H37Ra ( C ) at MOI 10. The levels of pro9 caspase 8 and RIPK3 were determined by subjecting equal amounts of mitochondria isolated from infected Mφ to Western blotting. VDAC was used as a loading control. ( D ) Colocalization of caspase 8 and RIPK3 with the mitochondria of H37Rv-infected Mφ. Left panels: Colocalization of RIPK3 and pro-caspase 8 with mitochondria 24h after infection visualized by confocal fluorescence microscopy. Human Mφ remained either uninfected or were infected with mCherry- H37Rv (MOI 10) for 24h, fixed and stained with mitotracker (green) and anti-RIPK3 or anti caspase 8 ab (red). Scale bar, 30 μm. On the right side of every panel is the quantification of caspase 8 (top) and of RIPK3 (bottom) associated with the mitochondria. Data were analyzed using nonparametric Student t test ( E ) Phase-contrast images of representative infected and uninfected human primary Mφ after 24 h of infection show approximate location of the plasma membrane and the nucleus as indicated by black and white arrows, respectively. Due to incipient necrosis the plasma membrane of the H37Rv-infected Mφ is not clearly visible. ( F ) BMD-Mφ from WT and RIPK3 −/− mice were infected with H37Rv at an MOI of ~10. After 12 h, expression of cleaved caspase 8 was assessed using flowcytometry. Results are represented as mean ± SD. Data were analyzed using one-way ANOVA. *,**,*** Values of P < 0.05, P <0.01 and P<0.001 , respectively were considered to be significant. Data are representative of 2–3 independent experiments.

    Techniques Used: Infection, Isolation, Immunoprecipitation, Western Blot, Translocation Assay, Fluorescence, Microscopy, Staining, Expressing

    ( A ) Bcl-x L is required for pro-caspase 8 and RIPK3 accumulation on the mitochondria of H37Rv infected Mφ. Human Mφ were transfected with Bcl-x L or scrambled control (Scr) siRNA and then infected with H37Rv (MOI 10). Equal amounts of purified mitochondria from non-infected and H37Rv-infected Mφ were subjected to Western blot analysis to determine the levels of pro-caspase 8 and RIPK3. ( B ) Caspase inhibition of H37Ra infected Mφ enables RIPK3/Bcl-x L accumulation on mitochondria. After treatment with the caspase inhibitor z-IETD pro-caspase 8, RIPK3 and Bcl-x L accumulates on the mitochondria of H37Ra infected Mφ. Mitochondria were isolated from H37Rv or H37Ra-infected Mφ treated with or without z-IETD (10 μmol) and subjected to Western blot analysis using anti caspase 8, anti-RIPK3 and anti-Bcl-x L ab. Equal amounts of the proteins were subjected to Western blot analysis for the evaluation of RIPK3 and Bcl-x L levels. ( C ) Mφ treated with Bcl-x L or scrambled control siRNA (Scr) were infected with H37Rv (MOI 10) and cell death was assessed after 48 h using Live/Dead fixable dead cell stain kits (Invitrogen). (D) z-IETD blocks activation of the apoptotic caspase 9 and 3 essential for apoptosis induction. Equal amounts of cell lysates of Mφ infected with H37Ra (pro-apoptotic strain) treated with or without z-IETD (10 μmol) were subjected to Western blot analysis for evaluating active caspase 3 and 9. ( E ) RIPK3 is required for accumulation of mitochondrial pro-caspase 8 and Bcl-x L . Mitochondria and cytosolic fraction of H37Rv-infected Mφ treated with RIPK3 or Scr siRNA were subjected to Western blot analysis and the levels of RIPK3, pro-caspase 8 and Bcl-x L were evaluated. (F ) Silencing of the RIPK3 gene activates apoptosis executor caspase 3 in H37Rv infected Mφ. Mφ treated with RIPK3 or Scr siRNA were infected with H37Rv. Cell lysate was collected and equal amounts subjected to Western blot analysis for cleaved caspase 3. ( G ) Bid processing in H37Ra and H37Rv-infected Mφ. Mitochondria were isolated from H37Ra (right panel) or H37Rv (left panel) -infected Mφ and the kinetics of BID processing and tBID accumulation were assessed by Western blotting. ( H ) Silencing of the BAX gene in H37Ra-infected Mφ blocks caspase 3 activation, a marker for apoptosis. Mφ treated with BAX or Scr siRNA were infected with H37Ra or H37Rv (MOI 10). Cell lysate was collected after 0 and 24 h and equal amounts subjected to Western blot analysis for pro-caspase 3 and cleaved caspase 3. VDAC and GAPDH were used as a loading control. Results are expressed as mean ± SD. Data were analyzed using one-way ANOVA. *, Values of P < 0.05 were considered to be significant. Data are representative of four independent experiments.
    Figure Legend Snippet: ( A ) Bcl-x L is required for pro-caspase 8 and RIPK3 accumulation on the mitochondria of H37Rv infected Mφ. Human Mφ were transfected with Bcl-x L or scrambled control (Scr) siRNA and then infected with H37Rv (MOI 10). Equal amounts of purified mitochondria from non-infected and H37Rv-infected Mφ were subjected to Western blot analysis to determine the levels of pro-caspase 8 and RIPK3. ( B ) Caspase inhibition of H37Ra infected Mφ enables RIPK3/Bcl-x L accumulation on mitochondria. After treatment with the caspase inhibitor z-IETD pro-caspase 8, RIPK3 and Bcl-x L accumulates on the mitochondria of H37Ra infected Mφ. Mitochondria were isolated from H37Rv or H37Ra-infected Mφ treated with or without z-IETD (10 μmol) and subjected to Western blot analysis using anti caspase 8, anti-RIPK3 and anti-Bcl-x L ab. Equal amounts of the proteins were subjected to Western blot analysis for the evaluation of RIPK3 and Bcl-x L levels. ( C ) Mφ treated with Bcl-x L or scrambled control siRNA (Scr) were infected with H37Rv (MOI 10) and cell death was assessed after 48 h using Live/Dead fixable dead cell stain kits (Invitrogen). (D) z-IETD blocks activation of the apoptotic caspase 9 and 3 essential for apoptosis induction. Equal amounts of cell lysates of Mφ infected with H37Ra (pro-apoptotic strain) treated with or without z-IETD (10 μmol) were subjected to Western blot analysis for evaluating active caspase 3 and 9. ( E ) RIPK3 is required for accumulation of mitochondrial pro-caspase 8 and Bcl-x L . Mitochondria and cytosolic fraction of H37Rv-infected Mφ treated with RIPK3 or Scr siRNA were subjected to Western blot analysis and the levels of RIPK3, pro-caspase 8 and Bcl-x L were evaluated. (F ) Silencing of the RIPK3 gene activates apoptosis executor caspase 3 in H37Rv infected Mφ. Mφ treated with RIPK3 or Scr siRNA were infected with H37Rv. Cell lysate was collected and equal amounts subjected to Western blot analysis for cleaved caspase 3. ( G ) Bid processing in H37Ra and H37Rv-infected Mφ. Mitochondria were isolated from H37Ra (right panel) or H37Rv (left panel) -infected Mφ and the kinetics of BID processing and tBID accumulation were assessed by Western blotting. ( H ) Silencing of the BAX gene in H37Ra-infected Mφ blocks caspase 3 activation, a marker for apoptosis. Mφ treated with BAX or Scr siRNA were infected with H37Ra or H37Rv (MOI 10). Cell lysate was collected after 0 and 24 h and equal amounts subjected to Western blot analysis for pro-caspase 3 and cleaved caspase 3. VDAC and GAPDH were used as a loading control. Results are expressed as mean ± SD. Data were analyzed using one-way ANOVA. *, Values of P < 0.05 were considered to be significant. Data are representative of four independent experiments.

    Techniques Used: Infection, Transfection, Purification, Western Blot, Inhibition, Isolation, Staining, Activation Assay, Marker

    ( A ) RIPK3 is required for MPT in H37Rv infected Mφ. Mφ were transfected with RIPK3 or scrambled (Scr) control siRNA and were then infected with H37Rv (MOI 10). Cationic dye (DiOC6 (3) ) release from mitochondria (a measure for MPT) was measured at 48 and 72 h after infection. ( B and C ) Inhibition of CypD reduces ROS-dependent necrosis. ( B ) Equal numbers of CsA-treated (5 μM) and untreated Mφ were infected with H37Rv (MOI 5 or 10). After 48 h of infection ROS accumulation ( B ) was measured by FACS analysis using the fluorescent dye CM-H 2 XRos and cell viability ( C ) was determined using the Live-Dead Assay. ( D ) CypD inactivation reduces translocation of RIPK3, pro-caspase 8, Bcl-x L and HKII to the mitochondria in H37Rv-infected Mφ at 24 h. Equal amounts of mitochondria from H37Rv-infected Mφ treated with or without CsA (5 μM) were subjected to Western blot analysis for RIPK3, pro-caspase 8, Bcl-x L and HKII after 24 h of infection. ( E ) Mitochondria from H37Ra or H37Rv infected Mφ were subjected to IP with anti-ANT ab and were then subjected to Western blot analysis for CypD. ( F ) CypD - ANT interaction is augmented in H37Rv-infected Mφ and is inhibited by inactivation of CypD with CsA (5 μM). ( G ) Top panel: RIPK3 is required for CypD - ANT interaction on the mitochondria. After 24h of infection, mitochondria from H37Rv-infected Mφ transfected with RIPK3 or scrambled (Scr) control siRNA were subjected to IP with anti-ANT ab and were then analyzed by Western blot for CypD. ANT was used as a loading control. Bottom panel: Silencing efficiency of RIPK3 siRNA. VDAC was used as a loading control. Results are expressed as mean ± SE, using nonparametric Student t test. *, Values of P < 0.05 were considered to be significant. Data are representative of three independent experiments.
    Figure Legend Snippet: ( A ) RIPK3 is required for MPT in H37Rv infected Mφ. Mφ were transfected with RIPK3 or scrambled (Scr) control siRNA and were then infected with H37Rv (MOI 10). Cationic dye (DiOC6 (3) ) release from mitochondria (a measure for MPT) was measured at 48 and 72 h after infection. ( B and C ) Inhibition of CypD reduces ROS-dependent necrosis. ( B ) Equal numbers of CsA-treated (5 μM) and untreated Mφ were infected with H37Rv (MOI 5 or 10). After 48 h of infection ROS accumulation ( B ) was measured by FACS analysis using the fluorescent dye CM-H 2 XRos and cell viability ( C ) was determined using the Live-Dead Assay. ( D ) CypD inactivation reduces translocation of RIPK3, pro-caspase 8, Bcl-x L and HKII to the mitochondria in H37Rv-infected Mφ at 24 h. Equal amounts of mitochondria from H37Rv-infected Mφ treated with or without CsA (5 μM) were subjected to Western blot analysis for RIPK3, pro-caspase 8, Bcl-x L and HKII after 24 h of infection. ( E ) Mitochondria from H37Ra or H37Rv infected Mφ were subjected to IP with anti-ANT ab and were then subjected to Western blot analysis for CypD. ( F ) CypD - ANT interaction is augmented in H37Rv-infected Mφ and is inhibited by inactivation of CypD with CsA (5 μM). ( G ) Top panel: RIPK3 is required for CypD - ANT interaction on the mitochondria. After 24h of infection, mitochondria from H37Rv-infected Mφ transfected with RIPK3 or scrambled (Scr) control siRNA were subjected to IP with anti-ANT ab and were then analyzed by Western blot for CypD. ANT was used as a loading control. Bottom panel: Silencing efficiency of RIPK3 siRNA. VDAC was used as a loading control. Results are expressed as mean ± SE, using nonparametric Student t test. *, Values of P < 0.05 were considered to be significant. Data are representative of three independent experiments.

    Techniques Used: Infection, Transfection, Inhibition, Live Dead Assay, Translocation Assay, Western Blot

    In Mφ infected with virulent Mtb RIPK3 and pro-caspase 8 present in the cytosol translocate to the mitochondria in presence of Bcl-x L and RIPK3 is activated RIPK3 enhances binding of HKII to VDAC on the outer mitochondrial membrane controlling mitochondrial glycolysis. At the same time activated RIPK3 triggers CypD-dependent formation of the mitochondrial permeability transition (MPT) pore via interaction between ANT and VDAC leading to leakage of the electron chain. Both mechanisms seem to be required for increasing ROS-dependent necrosis (right). In Mφ infected with avirulent Mtb the RIPK3 and caspase 8 also translocate to the mitochondria but this step is quickly followed by activation of caspase 8 and degradation of RIPK3. Oligomerization of BAX and BAK, which in turn allows the release of pro-apoptotic molecules (e.g. cytochrome c) leads to apoptosis (left). The exact action mechanism of Bcl-x L function is unknown.
    Figure Legend Snippet: In Mφ infected with virulent Mtb RIPK3 and pro-caspase 8 present in the cytosol translocate to the mitochondria in presence of Bcl-x L and RIPK3 is activated RIPK3 enhances binding of HKII to VDAC on the outer mitochondrial membrane controlling mitochondrial glycolysis. At the same time activated RIPK3 triggers CypD-dependent formation of the mitochondrial permeability transition (MPT) pore via interaction between ANT and VDAC leading to leakage of the electron chain. Both mechanisms seem to be required for increasing ROS-dependent necrosis (right). In Mφ infected with avirulent Mtb the RIPK3 and caspase 8 also translocate to the mitochondria but this step is quickly followed by activation of caspase 8 and degradation of RIPK3. Oligomerization of BAX and BAK, which in turn allows the release of pro-apoptotic molecules (e.g. cytochrome c) leads to apoptosis (left). The exact action mechanism of Bcl-x L function is unknown.

    Techniques Used: Infection, Binding Assay, Permeability, Activation Assay

    monoclonal rabbit anticleaved caspase 8 asp387 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit mab cleaved caspase 8 asp387
    NLRC4 regulates early IL-1β release after P. brasiliensis infection WT and Nlrc4 −/− and mice were intravenously infected with 1x10 6 of viable Pb18 yeasts. (A) Scatterplots with bars show the fungal load in lung of WT and Nlrc4 −/− mice at 7 dpi. Results represent a pool of three independent experiments. (B) Scatterplots with bars show the quantification of IL-1β at 7 in the lungs of WT and Nlrc4 −/− mice. (C) Western blotting analysis detecting IL-1β cleavage (p17) in lung homogenates from WT and Nlrc4 −/− mice at 7 dpi. The intensities of IL-1β protein were quantified using iBright™ CL1500 Imaging System. (D) IL-1β expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 50 μM – left panel). Each column represents the mean ± SD. (E) Caspase-1 expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 20 μM – right panel). Each column represents the mean ± SD. (F) Active caspase-1 (p20) was detected by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-1 were quantified using iBright™ CL1500 Imaging System. (G) Active <t>caspase-8</t> (p18) was detect by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-8 were quantified using iBright™ CL1500 Imaging System. (H) Stacked bars show the percentage of neutrophils (Ly6G + CD11b + ), monocytes (Ly6G − , Ly6C + CD11b + ), macrophages (CD11b + F4/80 + MHC-II + ), and dendritic cells (CD11c + CD11b + MHC-II + ). (I) Contour plots show representative flow cytometric data and scatterplots with bars show the absolute numbers of macrophages and dendritic cells gated in CD45 + IL-1β + cells at 7 dpi (n = 5 infected mice per group at 7 dpi).Data represent one of two independent experiments ( n = 3– 5 infected mice per group at each time point). Data are expressed as mean ± SD. Statistically significant differences were evaluated with ANOVA followed by Bonferroni's multiple comparisons test or unpaired t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also <xref ref-type=Figure S1 . " width="250" height="auto" />
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    Reduced DNA Damage And Genetic Instability in Mcl-1 Δhep /TNFR1 −/− and <t>TAK1/Casp8</t> Δhep Mice and Intercrossings (A) Staining for γH2AX (black) and cleaved Casp3 (red), double-positive hepatocytes (black/red arrows). Scale bar, 50 μm. (B) IF staining for γH2AX and Ki67 in wild-type, Mcl-1 Δhep , and Mcl-1 Δhep /TNFR1 −/− mice, as well as TAK1 Δhep , TAK1/Casp8 Δhep , and TAK1 Δhep /RIPK3 −/− mice. Arrowheads indicate cells with positive IF staining. Scale bar, 10 μm. (C) Quantification of Ki67 + and Ki67 + /γH2AX + hepatocytes (n = 4 mice per group, n = 5 for Mcl-1 Δhep mice). (D) Rate of AI in wild-type, Mcl-1 Δhep , and Mcl-1 Δhep /TNFR1 −/− mice, TAK1 Δhep , TAK1 Δhep /RIPK3 −/− , and TAK1/Casp8 Δhep mice (TaqMan copy number assay, each square represents one area of microdissected liver tissue, lines indicate different areas of the same liver; red, AI; black, no AI). Mcl-1 Δhep mice and intercrossings at 2 months; TAK1 Δhep mice and intercrossings at 6 weeks of age. In (C), data are presented as mean ± SEM. Statistical significance was calculated using ANOVA with Bonferroni correction (C), or Fisher's exact test (D). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also <xref ref-type=Figure S5 . " width="250" height="auto" />
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    ( A ) Cytosolic and mitochondrial RIPK3 and pro3 <t>caspase-8</t> following H37Rv infection. Equal numbers of human Mφ were infected with H37Rv (MOI 10) and lysed for isolation of cytosolic and mitochondrial fractions after 24 h of infection. Equal amounts of protein were immunoprecipitated (IP) with anti-caspase-8 ab and the levels of RIPK3, RIPK1, and pro-caspase 8 were measured by Western blot analysis. ( B-C ) Time7 dependent translocation of RIPK3 and pro-caspase 8 to the mitochondria. Equal numbers of Mφ were infected with virulent H37Rv or ( B ) avirulent H37Ra ( C ) at MOI 10. The levels of pro9 caspase 8 and RIPK3 were determined by subjecting equal amounts of mitochondria isolated from infected Mφ to Western blotting. VDAC was used as a loading control. ( D ) Colocalization of caspase 8 and RIPK3 with the mitochondria of H37Rv-infected Mφ. Left panels: Colocalization of RIPK3 and pro-caspase 8 with mitochondria 24h after infection visualized by confocal fluorescence microscopy. Human Mφ remained either uninfected or were infected with mCherry- H37Rv (MOI 10) for 24h, fixed and stained with mitotracker (green) and anti-RIPK3 or anti caspase 8 ab (red). Scale bar, 30 μm. On the right side of every panel is the quantification of caspase 8 (top) and of RIPK3 (bottom) associated with the mitochondria. Data were analyzed using nonparametric Student t test ( E ) Phase-contrast images of representative infected and uninfected human primary Mφ after 24 h of infection show approximate location of the plasma membrane and the nucleus as indicated by black and white arrows, respectively. Due to incipient necrosis the plasma membrane of the H37Rv-infected Mφ is not clearly visible. ( F ) BMD-Mφ from WT and RIPK3 −/− mice were infected with H37Rv at an MOI of ~10. After 12 h, expression of cleaved caspase 8 was assessed using flowcytometry. Results are represented as mean ± SD. Data were analyzed using one-way ANOVA. *,**,*** Values of P < 0.05, P <0.01 and P<0.001 , respectively were considered to be significant. Data are representative of 2–3 independent experiments.
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    ( A ) Cytosolic and mitochondrial RIPK3 and pro3 <t>caspase-8</t> following H37Rv infection. Equal numbers of human Mφ were infected with H37Rv (MOI 10) and lysed for isolation of cytosolic and mitochondrial fractions after 24 h of infection. Equal amounts of protein were immunoprecipitated (IP) with anti-caspase-8 ab and the levels of RIPK3, RIPK1, and pro-caspase 8 were measured by Western blot analysis. ( B-C ) Time7 dependent translocation of RIPK3 and pro-caspase 8 to the mitochondria. Equal numbers of Mφ were infected with virulent H37Rv or ( B ) avirulent H37Ra ( C ) at MOI 10. The levels of pro9 caspase 8 and RIPK3 were determined by subjecting equal amounts of mitochondria isolated from infected Mφ to Western blotting. VDAC was used as a loading control. ( D ) Colocalization of caspase 8 and RIPK3 with the mitochondria of H37Rv-infected Mφ. Left panels: Colocalization of RIPK3 and pro-caspase 8 with mitochondria 24h after infection visualized by confocal fluorescence microscopy. Human Mφ remained either uninfected or were infected with mCherry- H37Rv (MOI 10) for 24h, fixed and stained with mitotracker (green) and anti-RIPK3 or anti caspase 8 ab (red). Scale bar, 30 μm. On the right side of every panel is the quantification of caspase 8 (top) and of RIPK3 (bottom) associated with the mitochondria. Data were analyzed using nonparametric Student t test ( E ) Phase-contrast images of representative infected and uninfected human primary Mφ after 24 h of infection show approximate location of the plasma membrane and the nucleus as indicated by black and white arrows, respectively. Due to incipient necrosis the plasma membrane of the H37Rv-infected Mφ is not clearly visible. ( F ) BMD-Mφ from WT and RIPK3 −/− mice were infected with H37Rv at an MOI of ~10. After 12 h, expression of cleaved caspase 8 was assessed using flowcytometry. Results are represented as mean ± SD. Data were analyzed using one-way ANOVA. *,**,*** Values of P < 0.05, P <0.01 and P<0.001 , respectively were considered to be significant. Data are representative of 2–3 independent experiments.
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    ( A ) Cytosolic and mitochondrial RIPK3 and pro3 <t>caspase-8</t> following H37Rv infection. Equal numbers of human Mφ were infected with H37Rv (MOI 10) and lysed for isolation of cytosolic and mitochondrial fractions after 24 h of infection. Equal amounts of protein were immunoprecipitated (IP) with anti-caspase-8 ab and the levels of RIPK3, RIPK1, and pro-caspase 8 were measured by Western blot analysis. ( B-C ) Time7 dependent translocation of RIPK3 and pro-caspase 8 to the mitochondria. Equal numbers of Mφ were infected with virulent H37Rv or ( B ) avirulent H37Ra ( C ) at MOI 10. The levels of pro9 caspase 8 and RIPK3 were determined by subjecting equal amounts of mitochondria isolated from infected Mφ to Western blotting. VDAC was used as a loading control. ( D ) Colocalization of caspase 8 and RIPK3 with the mitochondria of H37Rv-infected Mφ. Left panels: Colocalization of RIPK3 and pro-caspase 8 with mitochondria 24h after infection visualized by confocal fluorescence microscopy. Human Mφ remained either uninfected or were infected with mCherry- H37Rv (MOI 10) for 24h, fixed and stained with mitotracker (green) and anti-RIPK3 or anti caspase 8 ab (red). Scale bar, 30 μm. On the right side of every panel is the quantification of caspase 8 (top) and of RIPK3 (bottom) associated with the mitochondria. Data were analyzed using nonparametric Student t test ( E ) Phase-contrast images of representative infected and uninfected human primary Mφ after 24 h of infection show approximate location of the plasma membrane and the nucleus as indicated by black and white arrows, respectively. Due to incipient necrosis the plasma membrane of the H37Rv-infected Mφ is not clearly visible. ( F ) BMD-Mφ from WT and RIPK3 −/− mice were infected with H37Rv at an MOI of ~10. After 12 h, expression of cleaved caspase 8 was assessed using flowcytometry. Results are represented as mean ± SD. Data were analyzed using one-way ANOVA. *,**,*** Values of P < 0.05, P <0.01 and P<0.001 , respectively were considered to be significant. Data are representative of 2–3 independent experiments.
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    ( A ) Cytosolic and mitochondrial RIPK3 and pro3 <t>caspase-8</t> following H37Rv infection. Equal numbers of human Mφ were infected with H37Rv (MOI 10) and lysed for isolation of cytosolic and mitochondrial fractions after 24 h of infection. Equal amounts of protein were immunoprecipitated (IP) with anti-caspase-8 ab and the levels of RIPK3, RIPK1, and pro-caspase 8 were measured by Western blot analysis. ( B-C ) Time7 dependent translocation of RIPK3 and pro-caspase 8 to the mitochondria. Equal numbers of Mφ were infected with virulent H37Rv or ( B ) avirulent H37Ra ( C ) at MOI 10. The levels of pro9 caspase 8 and RIPK3 were determined by subjecting equal amounts of mitochondria isolated from infected Mφ to Western blotting. VDAC was used as a loading control. ( D ) Colocalization of caspase 8 and RIPK3 with the mitochondria of H37Rv-infected Mφ. Left panels: Colocalization of RIPK3 and pro-caspase 8 with mitochondria 24h after infection visualized by confocal fluorescence microscopy. Human Mφ remained either uninfected or were infected with mCherry- H37Rv (MOI 10) for 24h, fixed and stained with mitotracker (green) and anti-RIPK3 or anti caspase 8 ab (red). Scale bar, 30 μm. On the right side of every panel is the quantification of caspase 8 (top) and of RIPK3 (bottom) associated with the mitochondria. Data were analyzed using nonparametric Student t test ( E ) Phase-contrast images of representative infected and uninfected human primary Mφ after 24 h of infection show approximate location of the plasma membrane and the nucleus as indicated by black and white arrows, respectively. Due to incipient necrosis the plasma membrane of the H37Rv-infected Mφ is not clearly visible. ( F ) BMD-Mφ from WT and RIPK3 −/− mice were infected with H37Rv at an MOI of ~10. After 12 h, expression of cleaved caspase 8 was assessed using flowcytometry. Results are represented as mean ± SD. Data were analyzed using one-way ANOVA. *,**,*** Values of P < 0.05, P <0.01 and P<0.001 , respectively were considered to be significant. Data are representative of 2–3 independent experiments.
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    NLRC4 regulates early IL-1β release after P. brasiliensis infection WT and Nlrc4 −/− and mice were intravenously infected with 1x10 6 of viable Pb18 yeasts. (A) Scatterplots with bars show the fungal load in lung of WT and Nlrc4 −/− mice at 7 dpi. Results represent a pool of three independent experiments. (B) Scatterplots with bars show the quantification of IL-1β at 7 in the lungs of WT and Nlrc4 −/− mice. (C) Western blotting analysis detecting IL-1β cleavage (p17) in lung homogenates from WT and Nlrc4 −/− mice at 7 dpi. The intensities of IL-1β protein were quantified using iBright™ CL1500 Imaging System. (D) IL-1β expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 50 μM – left panel). Each column represents the mean ± SD. (E) Caspase-1 expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 20 μM – right panel). Each column represents the mean ± SD. (F) Active caspase-1 (p20) was detected by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-1 were quantified using iBright™ CL1500 Imaging System. (G) Active caspase-8 (p18) was detect by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-8 were quantified using iBright™ CL1500 Imaging System. (H) Stacked bars show the percentage of neutrophils (Ly6G + CD11b + ), monocytes (Ly6G − , Ly6C + CD11b + ), macrophages (CD11b + F4/80 + MHC-II + ), and dendritic cells (CD11c + CD11b + MHC-II + ). (I) Contour plots show representative flow cytometric data and scatterplots with bars show the absolute numbers of macrophages and dendritic cells gated in CD45 + IL-1β + cells at 7 dpi (n = 5 infected mice per group at 7 dpi).Data represent one of two independent experiments ( n = 3– 5 infected mice per group at each time point). Data are expressed as mean ± SD. Statistically significant differences were evaluated with ANOVA followed by Bonferroni's multiple comparisons test or unpaired t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also <xref ref-type=Figure S1 . " width="100%" height="100%">

    Journal: iScience

    Article Title: NLRC4 inhibits NLRP3 inflammasome and abrogates effective antifungal CD8 + T cell responses

    doi: 10.1016/j.isci.2021.102548

    Figure Lengend Snippet: NLRC4 regulates early IL-1β release after P. brasiliensis infection WT and Nlrc4 −/− and mice were intravenously infected with 1x10 6 of viable Pb18 yeasts. (A) Scatterplots with bars show the fungal load in lung of WT and Nlrc4 −/− mice at 7 dpi. Results represent a pool of three independent experiments. (B) Scatterplots with bars show the quantification of IL-1β at 7 in the lungs of WT and Nlrc4 −/− mice. (C) Western blotting analysis detecting IL-1β cleavage (p17) in lung homogenates from WT and Nlrc4 −/− mice at 7 dpi. The intensities of IL-1β protein were quantified using iBright™ CL1500 Imaging System. (D) IL-1β expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 50 μM – left panel). Each column represents the mean ± SD. (E) Caspase-1 expression was visualized in the lungs of mice at 7 dpi from both groups using immunohistochemistry (Scale bar: 20 μM – right panel). Each column represents the mean ± SD. (F) Active caspase-1 (p20) was detected by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-1 were quantified using iBright™ CL1500 Imaging System. (G) Active caspase-8 (p18) was detect by Western blotting in the lungs from WT and Nlc4 −/− mice at 7 dpi. The intensities of active caspase-8 were quantified using iBright™ CL1500 Imaging System. (H) Stacked bars show the percentage of neutrophils (Ly6G + CD11b + ), monocytes (Ly6G − , Ly6C + CD11b + ), macrophages (CD11b + F4/80 + MHC-II + ), and dendritic cells (CD11c + CD11b + MHC-II + ). (I) Contour plots show representative flow cytometric data and scatterplots with bars show the absolute numbers of macrophages and dendritic cells gated in CD45 + IL-1β + cells at 7 dpi (n = 5 infected mice per group at 7 dpi).Data represent one of two independent experiments ( n = 3– 5 infected mice per group at each time point). Data are expressed as mean ± SD. Statistically significant differences were evaluated with ANOVA followed by Bonferroni's multiple comparisons test or unpaired t-test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). See also Figure S1 .

    Article Snippet: Rabbit mAb cleaved Caspase-8 (Asp387) (D5B2) XP® , Cell Signaling Technology , Cat#8592S; RRID: AB_10891784.

    Techniques: Infection, Western Blot, Imaging, Expressing, Immunohistochemistry

    Journal: iScience

    Article Title: NLRC4 inhibits NLRP3 inflammasome and abrogates effective antifungal CD8 + T cell responses

    doi: 10.1016/j.isci.2021.102548

    Figure Lengend Snippet:

    Article Snippet: Rabbit mAb cleaved Caspase-8 (Asp387) (D5B2) XP® , Cell Signaling Technology , Cat#8592S; RRID: AB_10891784.

    Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Bradford Protein Assay, Knock-Out, Double Knockout, Mouse Assay, Software, Imaging

    Antibodies

    Journal: Cellular and Molecular Neurobiology

    Article Title: TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection

    doi: 10.1007/s10571-021-01063-w

    Figure Lengend Snippet: Antibodies

    Article Snippet: Cleaved caspase-8 (Asp387) (D5B2) XP® rabbit mAb (mouse specific) , Cell signaling technology , #8592.

    Techniques:

    The PPI network for the core enriched genes related to the apoptosis and necroptosis pathways. a , b . PPI network analysis for the core enriched genes of GSEA (as shown in Fig. c, d) showed that caspase-8 and TNF-α were the hub genes for the apoptosis and necroptosis pathways in mouse brains infected by AC, respectively. AC Angiostrongylus cantonensis

    Journal: Cellular and Molecular Neurobiology

    Article Title: TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection

    doi: 10.1007/s10571-021-01063-w

    Figure Lengend Snippet: The PPI network for the core enriched genes related to the apoptosis and necroptosis pathways. a , b . PPI network analysis for the core enriched genes of GSEA (as shown in Fig. c, d) showed that caspase-8 and TNF-α were the hub genes for the apoptosis and necroptosis pathways in mouse brains infected by AC, respectively. AC Angiostrongylus cantonensis

    Article Snippet: Cleaved caspase-8 (Asp387) (D5B2) XP® rabbit mAb (mouse specific) , Cell signaling technology , #8592.

    Techniques: Infection

    TNF-α triggers RIP1/FADD/caspase-8-mediated apoptosis of astrocytes in mouse brains with AC infection. a Relative mRNA level of TNF-α in mouse brains upon AC infection was significantly higher than that in the control group. b TNF-α protein expression level in the mouse brain post infection was remarkably elevated, as revealed by immunofluorescence (left) and relative mean fluorescence intensity (MFI, right). c The lysates of brain tissues from normal control mice or mice post infection were subjected to western blot to determine the protein levels of genes related to the apoptosis signalling pathway (TNF-α, RIP1, caspase-8 and cleaved caspase-8). d – g The protein expression levels in c were quantified via density analysis. h Mice were infected by AC for 21 days and then the lysates of mouse brain tissues were immunoprecipitated with an anti-RIP1 antibody or an IgG antibody and the precipitated complexes were separately analysed by immunoblotting with antibodies against RIP1, RIP3, FADD, caspase-8 and GAPDH. i Cleaved caspase-3 and GFAP (specific marker of astrocytes) were co-localized, as shown by immunofluorescence. j Cleaved caspase-3 and NeuN (specific marker of neurons) showed no co-localization, as shown by immunofluorescence. * p < 0.05, ** p < 0.01, *** p < 0.001 (student’s t test). AC Angiostrongylus cantonensis , Ctrl normal control, INF infected by Angiostrongylus cantonensis

    Journal: Cellular and Molecular Neurobiology

    Article Title: TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection

    doi: 10.1007/s10571-021-01063-w

    Figure Lengend Snippet: TNF-α triggers RIP1/FADD/caspase-8-mediated apoptosis of astrocytes in mouse brains with AC infection. a Relative mRNA level of TNF-α in mouse brains upon AC infection was significantly higher than that in the control group. b TNF-α protein expression level in the mouse brain post infection was remarkably elevated, as revealed by immunofluorescence (left) and relative mean fluorescence intensity (MFI, right). c The lysates of brain tissues from normal control mice or mice post infection were subjected to western blot to determine the protein levels of genes related to the apoptosis signalling pathway (TNF-α, RIP1, caspase-8 and cleaved caspase-8). d – g The protein expression levels in c were quantified via density analysis. h Mice were infected by AC for 21 days and then the lysates of mouse brain tissues were immunoprecipitated with an anti-RIP1 antibody or an IgG antibody and the precipitated complexes were separately analysed by immunoblotting with antibodies against RIP1, RIP3, FADD, caspase-8 and GAPDH. i Cleaved caspase-3 and GFAP (specific marker of astrocytes) were co-localized, as shown by immunofluorescence. j Cleaved caspase-3 and NeuN (specific marker of neurons) showed no co-localization, as shown by immunofluorescence. * p < 0.05, ** p < 0.01, *** p < 0.001 (student’s t test). AC Angiostrongylus cantonensis , Ctrl normal control, INF infected by Angiostrongylus cantonensis

    Article Snippet: Cleaved caspase-8 (Asp387) (D5B2) XP® rabbit mAb (mouse specific) , Cell signaling technology , #8592.

    Techniques: Infection, Expressing, Immunofluorescence, Fluorescence, Western Blot, Immunoprecipitation, Marker

    Reduced DNA Damage And Genetic Instability in Mcl-1 Δhep /TNFR1 −/− and TAK1/Casp8 Δhep Mice and Intercrossings (A) Staining for γH2AX (black) and cleaved Casp3 (red), double-positive hepatocytes (black/red arrows). Scale bar, 50 μm. (B) IF staining for γH2AX and Ki67 in wild-type, Mcl-1 Δhep , and Mcl-1 Δhep /TNFR1 −/− mice, as well as TAK1 Δhep , TAK1/Casp8 Δhep , and TAK1 Δhep /RIPK3 −/− mice. Arrowheads indicate cells with positive IF staining. Scale bar, 10 μm. (C) Quantification of Ki67 + and Ki67 + /γH2AX + hepatocytes (n = 4 mice per group, n = 5 for Mcl-1 Δhep mice). (D) Rate of AI in wild-type, Mcl-1 Δhep , and Mcl-1 Δhep /TNFR1 −/− mice, TAK1 Δhep , TAK1 Δhep /RIPK3 −/− , and TAK1/Casp8 Δhep mice (TaqMan copy number assay, each square represents one area of microdissected liver tissue, lines indicate different areas of the same liver; red, AI; black, no AI). Mcl-1 Δhep mice and intercrossings at 2 months; TAK1 Δhep mice and intercrossings at 6 weeks of age. In (C), data are presented as mean ± SEM. Statistical significance was calculated using ANOVA with Bonferroni correction (C), or Fisher's exact test (D). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also <xref ref-type=Figure S5 . " width="100%" height="100%">

    Journal: Cancer Cell

    Article Title: A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development

    doi: 10.1016/j.ccell.2017.08.010

    Figure Lengend Snippet: Reduced DNA Damage And Genetic Instability in Mcl-1 Δhep /TNFR1 −/− and TAK1/Casp8 Δhep Mice and Intercrossings (A) Staining for γH2AX (black) and cleaved Casp3 (red), double-positive hepatocytes (black/red arrows). Scale bar, 50 μm. (B) IF staining for γH2AX and Ki67 in wild-type, Mcl-1 Δhep , and Mcl-1 Δhep /TNFR1 −/− mice, as well as TAK1 Δhep , TAK1/Casp8 Δhep , and TAK1 Δhep /RIPK3 −/− mice. Arrowheads indicate cells with positive IF staining. Scale bar, 10 μm. (C) Quantification of Ki67 + and Ki67 + /γH2AX + hepatocytes (n = 4 mice per group, n = 5 for Mcl-1 Δhep mice). (D) Rate of AI in wild-type, Mcl-1 Δhep , and Mcl-1 Δhep /TNFR1 −/− mice, TAK1 Δhep , TAK1 Δhep /RIPK3 −/− , and TAK1/Casp8 Δhep mice (TaqMan copy number assay, each square represents one area of microdissected liver tissue, lines indicate different areas of the same liver; red, AI; black, no AI). Mcl-1 Δhep mice and intercrossings at 2 months; TAK1 Δhep mice and intercrossings at 6 weeks of age. In (C), data are presented as mean ± SEM. Statistical significance was calculated using ANOVA with Bonferroni correction (C), or Fisher's exact test (D). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. See also Figure S5 .

    Article Snippet: Cleaved Caspase-8 (Asp387) (D5B2) XP Rabbit mAb , Cell Signaling Technology , Cat# 8592.

    Techniques: Staining, TaqMan Copy Number Assay

    Detection of Proliferation-Associated DNA Damage after PHX Is Impaired in Casp8 Δhep Mice (A–C) Western blot analysis of whole-liver lysates (A), immunostainings (B), and quantification of γH2AX + hepatocytes 0, 6, 24, and 48 hr post-PHX (C). Scale bar, 50 μm. (D and E) BrdU incorporation combined with γH2AX staining (n = 4). Scale bar, 10 μm. (F and G) PFGE with densitometric quantification to visualize DNA DSB in livers of wild-type mice after PHX (n = 3). (H and I) IF staining (H) and quantification of Ki67 + /γH2AX + hepatocytes in wild-type, TNFR1/2 −/− , RIPK3 −/− , and Casp8 Δhep mice (I). Arrowheads indicate cells with positive IF staining. Scale bar, 10 μm. (J and K) PFGE with densitometric quantification to visualize DNA DSB in livers of Casp8 Δhep mice after PHX. In (C), bar represents mean. In (E), (G), (I), and (K) data are presented as mean ± SEM. In (G), bar indicates the mean. Statistical significance was calculated using ANOVA with Bonferroni correction (C and I) or Student's t test (E, G, and K). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s., not significant. Irrelevant bands were omitted from gels (F and J). Areas in which lanes were omitted are indicated by white space between lanes. See also <xref ref-type=Figure S6 . " width="100%" height="100%">

    Journal: Cancer Cell

    Article Title: A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development

    doi: 10.1016/j.ccell.2017.08.010

    Figure Lengend Snippet: Detection of Proliferation-Associated DNA Damage after PHX Is Impaired in Casp8 Δhep Mice (A–C) Western blot analysis of whole-liver lysates (A), immunostainings (B), and quantification of γH2AX + hepatocytes 0, 6, 24, and 48 hr post-PHX (C). Scale bar, 50 μm. (D and E) BrdU incorporation combined with γH2AX staining (n = 4). Scale bar, 10 μm. (F and G) PFGE with densitometric quantification to visualize DNA DSB in livers of wild-type mice after PHX (n = 3). (H and I) IF staining (H) and quantification of Ki67 + /γH2AX + hepatocytes in wild-type, TNFR1/2 −/− , RIPK3 −/− , and Casp8 Δhep mice (I). Arrowheads indicate cells with positive IF staining. Scale bar, 10 μm. (J and K) PFGE with densitometric quantification to visualize DNA DSB in livers of Casp8 Δhep mice after PHX. In (C), bar represents mean. In (E), (G), (I), and (K) data are presented as mean ± SEM. In (G), bar indicates the mean. Statistical significance was calculated using ANOVA with Bonferroni correction (C and I) or Student's t test (E, G, and K). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; n.s., not significant. Irrelevant bands were omitted from gels (F and J). Areas in which lanes were omitted are indicated by white space between lanes. See also Figure S6 .

    Article Snippet: Cleaved Caspase-8 (Asp387) (D5B2) XP Rabbit mAb , Cell Signaling Technology , Cat# 8592.

    Techniques: Western Blot, BrdU Incorporation Assay, Staining

    Caspase-8, RIPK1, FADD, and c-FLIP Are Crucial for Phosphorylation of H2AX in Hepatocytes upon Doxorubicin Treatment (A) IF for γH2AX in untreated wild-type mice and wild-type, Casp8 Δhep , and QVD-OPH-treated wild-type mice following doxorubicin treatment. Arrow heads illustrate γH2AX + foci in nuclei. Scale bar, 10 μm. (B) PFGE on livers of doxorubicin-treated mice. (C) γH2AX staining of doxorubicin-treated wild-type and caspase-8 D387-mutant mice. Scale bar, 50 μm. (D) γH2AX IF staining 12 hr post-doxorubicin-induced DNA damage in hepatocytes of Casp8 −/− /RIPK3 −/− mice (n = 5), RIPK3 −/− mice (n = 4), RIPK1 KD mice (n = 9), RIPK1 −/− RIPK3 −/− FADD −/− (labeled as R1 −/− R3 −/− FADD −/− , n = 2), RIPK1 +/− RIPK3 −/− FADD −/− (labeled as R1 +/− R3 −/− FADD −/− , n = 2), c-FLIP Δhep (n = 6), and TNFR1/2 −/− mice (n = 6). Arrowheads illustrate γH2AX + foci in nuclei. Scale bar, 10 μm. (E) Quantification of IF stainings (A and D). (F) Immunoprecipitation with anti-caspase-8 antibody (upper panel) and immunoblotting of lysates (lower panel), 0–24 hr after doxorubicin (5 μM) treatment. Red box: RIPK1, FADD, and caspase-8 interaction at 1 hr; blue boxes: low-level activation of apoptosis starting at 4 hr post-treatment. (The signal visible in the t = 0 column, cl.PARP lane, does not originate from cl.PARP, but from a lower unspecific band.) Control cells treated for 1 hr with CD95L/FasL (B, beads; L, lysates). (G) Immunoblotting of lysates, 0–24 hr after doxorubicin (5 μM) treatment looking at levels of total and cl.PARP, blue boxes (F and G): low-level activation of apoptosis starting at 4 hr post-treatment. (H) Levels of LUBAC (HOIP, HOIL-1, and SHARPIN), cIAP1, cIAP2, and XIAP in U2OS cells at 15 min (red box) post-doxorubicin stimulation (5 μM). (I) Subcellular fractionation of U2OS cells. (J) RIPK1 and γH2AX IF staining in U2OS cells after doxorubicin treatment. The arrowhead indicates colocalizing signals. Scale bar, 10 μm. Statistical significance was calculated using ANOVA with Bonferroni correction (E). ∗∗∗ p < 0.001. Irrelevant bands were omitted from gels (B). Areas in which lanes were omitted are indicated by white space between lanes. See also <xref ref-type=Figure S7 . " width="100%" height="100%">

    Journal: Cancer Cell

    Article Title: A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development

    doi: 10.1016/j.ccell.2017.08.010

    Figure Lengend Snippet: Caspase-8, RIPK1, FADD, and c-FLIP Are Crucial for Phosphorylation of H2AX in Hepatocytes upon Doxorubicin Treatment (A) IF for γH2AX in untreated wild-type mice and wild-type, Casp8 Δhep , and QVD-OPH-treated wild-type mice following doxorubicin treatment. Arrow heads illustrate γH2AX + foci in nuclei. Scale bar, 10 μm. (B) PFGE on livers of doxorubicin-treated mice. (C) γH2AX staining of doxorubicin-treated wild-type and caspase-8 D387-mutant mice. Scale bar, 50 μm. (D) γH2AX IF staining 12 hr post-doxorubicin-induced DNA damage in hepatocytes of Casp8 −/− /RIPK3 −/− mice (n = 5), RIPK3 −/− mice (n = 4), RIPK1 KD mice (n = 9), RIPK1 −/− RIPK3 −/− FADD −/− (labeled as R1 −/− R3 −/− FADD −/− , n = 2), RIPK1 +/− RIPK3 −/− FADD −/− (labeled as R1 +/− R3 −/− FADD −/− , n = 2), c-FLIP Δhep (n = 6), and TNFR1/2 −/− mice (n = 6). Arrowheads illustrate γH2AX + foci in nuclei. Scale bar, 10 μm. (E) Quantification of IF stainings (A and D). (F) Immunoprecipitation with anti-caspase-8 antibody (upper panel) and immunoblotting of lysates (lower panel), 0–24 hr after doxorubicin (5 μM) treatment. Red box: RIPK1, FADD, and caspase-8 interaction at 1 hr; blue boxes: low-level activation of apoptosis starting at 4 hr post-treatment. (The signal visible in the t = 0 column, cl.PARP lane, does not originate from cl.PARP, but from a lower unspecific band.) Control cells treated for 1 hr with CD95L/FasL (B, beads; L, lysates). (G) Immunoblotting of lysates, 0–24 hr after doxorubicin (5 μM) treatment looking at levels of total and cl.PARP, blue boxes (F and G): low-level activation of apoptosis starting at 4 hr post-treatment. (H) Levels of LUBAC (HOIP, HOIL-1, and SHARPIN), cIAP1, cIAP2, and XIAP in U2OS cells at 15 min (red box) post-doxorubicin stimulation (5 μM). (I) Subcellular fractionation of U2OS cells. (J) RIPK1 and γH2AX IF staining in U2OS cells after doxorubicin treatment. The arrowhead indicates colocalizing signals. Scale bar, 10 μm. Statistical significance was calculated using ANOVA with Bonferroni correction (E). ∗∗∗ p < 0.001. Irrelevant bands were omitted from gels (B). Areas in which lanes were omitted are indicated by white space between lanes. See also Figure S7 .

    Article Snippet: Cleaved Caspase-8 (Asp387) (D5B2) XP Rabbit mAb , Cell Signaling Technology , Cat# 8592.

    Techniques: Staining, Mutagenesis, Labeling, Immunoprecipitation, Western Blot, Activation Assay, Fractionation

    JNK Is a Downstream Mediator of Caspase-8-, c-FLIP-, and RIPK1-Dependent Phosphorylation of H2AX In Vivo and In Vitro (A) Immunohistochemistry for pCHK1, pCHK2, and pcJUN in livers after doxorubicin treatment. Arrowheads indicate pcJUN-positive nuclei. Scale bar, 50 μm. (B) γH2AX and pJNK co-stainings of livers 12 hr post-doxorubicin treatment. Merged: overlay of DAPI, γH2AX, and pJNK staining. Arrowheads indicate IF signals for γH2AX (green), pJNK (red), or overlapping signals of both (yellow). Scale bar, 10 μm. (C and D) IF stainings (C) and quantification for γH2AX in wild-type and JNK1/2-deficient hepatocytes 12 hr post-doxorubicin treatment (D). Arrowheads indicate IF signals for γH2AX. Scale bar, 10 μm. (E) Analysis of DDR signaling by western blotting of lysates from doxorubicin-treated caspase-8 knockdown cells, JNK inhibitor (SP600125) and ATM inhibitor (KU-55933) pre-treated control cells (U2OS). Red boxes: differences in γH2AX and pJNK activation post-doxorubicin treatment between control cells and lentiviral caspase-8 knockdown and JNK inhibitor treated cells. Statistical analysis was corrected for three tests using the Bonferroni method. See also <xref ref-type=Figure S8 . " width="100%" height="100%">

    Journal: Cancer Cell

    Article Title: A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development

    doi: 10.1016/j.ccell.2017.08.010

    Figure Lengend Snippet: JNK Is a Downstream Mediator of Caspase-8-, c-FLIP-, and RIPK1-Dependent Phosphorylation of H2AX In Vivo and In Vitro (A) Immunohistochemistry for pCHK1, pCHK2, and pcJUN in livers after doxorubicin treatment. Arrowheads indicate pcJUN-positive nuclei. Scale bar, 50 μm. (B) γH2AX and pJNK co-stainings of livers 12 hr post-doxorubicin treatment. Merged: overlay of DAPI, γH2AX, and pJNK staining. Arrowheads indicate IF signals for γH2AX (green), pJNK (red), or overlapping signals of both (yellow). Scale bar, 10 μm. (C and D) IF stainings (C) and quantification for γH2AX in wild-type and JNK1/2-deficient hepatocytes 12 hr post-doxorubicin treatment (D). Arrowheads indicate IF signals for γH2AX. Scale bar, 10 μm. (E) Analysis of DDR signaling by western blotting of lysates from doxorubicin-treated caspase-8 knockdown cells, JNK inhibitor (SP600125) and ATM inhibitor (KU-55933) pre-treated control cells (U2OS). Red boxes: differences in γH2AX and pJNK activation post-doxorubicin treatment between control cells and lentiviral caspase-8 knockdown and JNK inhibitor treated cells. Statistical analysis was corrected for three tests using the Bonferroni method. See also Figure S8 .

    Article Snippet: Cleaved Caspase-8 (Asp387) (D5B2) XP Rabbit mAb , Cell Signaling Technology , Cat# 8592.

    Techniques: In Vivo, In Vitro, Immunohistochemistry, Staining, Western Blot, Activation Assay

    Evidence for JNK-Dependent DDR in Human Regenerating Livers and Caspase-8 in Human HCC (A) γH2AX and pJNK co-stainings demonstrating JNK-dependent phosphorylation of H2AX in liver tissue of CLD patients or the left lobe of patients after (right) portal vein ligation and liver transection (PVL/LT). Arrowheads indicate IF signals for γH2AX (green), pJNK (red), or overlapping signals of both (yellow). Scale bar, 10 μm. (B) Overall survival of HCC patients depending on HCC caspase-8 expression level (<mean+1SD; n = 307 patients; >mean+1SD; n = 51 patients, log rank test, statistical analysis was corrected for three tests using the Bonferroni method. The Cancer Genome Atlas [TCGA] cohort). (C) Overall survival of HCC patients depending on HCC caspase-8 methylation status (n = 358 patients, TCGA cohort, log rank test).

    Journal: Cancer Cell

    Article Title: A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development

    doi: 10.1016/j.ccell.2017.08.010

    Figure Lengend Snippet: Evidence for JNK-Dependent DDR in Human Regenerating Livers and Caspase-8 in Human HCC (A) γH2AX and pJNK co-stainings demonstrating JNK-dependent phosphorylation of H2AX in liver tissue of CLD patients or the left lobe of patients after (right) portal vein ligation and liver transection (PVL/LT). Arrowheads indicate IF signals for γH2AX (green), pJNK (red), or overlapping signals of both (yellow). Scale bar, 10 μm. (B) Overall survival of HCC patients depending on HCC caspase-8 expression level (mean+1SD; n = 51 patients, log rank test, statistical analysis was corrected for three tests using the Bonferroni method. The Cancer Genome Atlas [TCGA] cohort). (C) Overall survival of HCC patients depending on HCC caspase-8 methylation status (n = 358 patients, TCGA cohort, log rank test).

    Article Snippet: Cleaved Caspase-8 (Asp387) (D5B2) XP Rabbit mAb , Cell Signaling Technology , Cat# 8592.

    Techniques: Ligation, Expressing, Methylation

    Journal: Cancer Cell

    Article Title: A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development

    doi: 10.1016/j.ccell.2017.08.010

    Figure Lengend Snippet:

    Article Snippet: Cleaved Caspase-8 (Asp387) (D5B2) XP Rabbit mAb , Cell Signaling Technology , Cat# 8592.

    Techniques: Purification, Imaging, Mutagenesis, Recombinant, Staining, SYBR Green Assay, Microarray, RNA Expression, RNA Sequencing Assay, Methylation, Labeling, Software, Light Microscopy

    ( A ) Cytosolic and mitochondrial RIPK3 and pro3 caspase-8 following H37Rv infection. Equal numbers of human Mφ were infected with H37Rv (MOI 10) and lysed for isolation of cytosolic and mitochondrial fractions after 24 h of infection. Equal amounts of protein were immunoprecipitated (IP) with anti-caspase-8 ab and the levels of RIPK3, RIPK1, and pro-caspase 8 were measured by Western blot analysis. ( B-C ) Time7 dependent translocation of RIPK3 and pro-caspase 8 to the mitochondria. Equal numbers of Mφ were infected with virulent H37Rv or ( B ) avirulent H37Ra ( C ) at MOI 10. The levels of pro9 caspase 8 and RIPK3 were determined by subjecting equal amounts of mitochondria isolated from infected Mφ to Western blotting. VDAC was used as a loading control. ( D ) Colocalization of caspase 8 and RIPK3 with the mitochondria of H37Rv-infected Mφ. Left panels: Colocalization of RIPK3 and pro-caspase 8 with mitochondria 24h after infection visualized by confocal fluorescence microscopy. Human Mφ remained either uninfected or were infected with mCherry- H37Rv (MOI 10) for 24h, fixed and stained with mitotracker (green) and anti-RIPK3 or anti caspase 8 ab (red). Scale bar, 30 μm. On the right side of every panel is the quantification of caspase 8 (top) and of RIPK3 (bottom) associated with the mitochondria. Data were analyzed using nonparametric Student t test ( E ) Phase-contrast images of representative infected and uninfected human primary Mφ after 24 h of infection show approximate location of the plasma membrane and the nucleus as indicated by black and white arrows, respectively. Due to incipient necrosis the plasma membrane of the H37Rv-infected Mφ is not clearly visible. ( F ) BMD-Mφ from WT and RIPK3 −/− mice were infected with H37Rv at an MOI of ~10. After 12 h, expression of cleaved caspase 8 was assessed using flowcytometry. Results are represented as mean ± SD. Data were analyzed using one-way ANOVA. *,**,*** Values of P < 0.05, P <0.01 and P<0.001 , respectively were considered to be significant. Data are representative of 2–3 independent experiments.

    Journal: Mucosal immunology

    Article Title: Bcl-x L mediates RIPK3-dependent necrosis in M. tuberculosis -infected macrophages

    doi: 10.1038/mi.2017.12

    Figure Lengend Snippet: ( A ) Cytosolic and mitochondrial RIPK3 and pro3 caspase-8 following H37Rv infection. Equal numbers of human Mφ were infected with H37Rv (MOI 10) and lysed for isolation of cytosolic and mitochondrial fractions after 24 h of infection. Equal amounts of protein were immunoprecipitated (IP) with anti-caspase-8 ab and the levels of RIPK3, RIPK1, and pro-caspase 8 were measured by Western blot analysis. ( B-C ) Time7 dependent translocation of RIPK3 and pro-caspase 8 to the mitochondria. Equal numbers of Mφ were infected with virulent H37Rv or ( B ) avirulent H37Ra ( C ) at MOI 10. The levels of pro9 caspase 8 and RIPK3 were determined by subjecting equal amounts of mitochondria isolated from infected Mφ to Western blotting. VDAC was used as a loading control. ( D ) Colocalization of caspase 8 and RIPK3 with the mitochondria of H37Rv-infected Mφ. Left panels: Colocalization of RIPK3 and pro-caspase 8 with mitochondria 24h after infection visualized by confocal fluorescence microscopy. Human Mφ remained either uninfected or were infected with mCherry- H37Rv (MOI 10) for 24h, fixed and stained with mitotracker (green) and anti-RIPK3 or anti caspase 8 ab (red). Scale bar, 30 μm. On the right side of every panel is the quantification of caspase 8 (top) and of RIPK3 (bottom) associated with the mitochondria. Data were analyzed using nonparametric Student t test ( E ) Phase-contrast images of representative infected and uninfected human primary Mφ after 24 h of infection show approximate location of the plasma membrane and the nucleus as indicated by black and white arrows, respectively. Due to incipient necrosis the plasma membrane of the H37Rv-infected Mφ is not clearly visible. ( F ) BMD-Mφ from WT and RIPK3 −/− mice were infected with H37Rv at an MOI of ~10. After 12 h, expression of cleaved caspase 8 was assessed using flowcytometry. Results are represented as mean ± SD. Data were analyzed using one-way ANOVA. *,**,*** Values of P < 0.05, P <0.01 and P<0.001 , respectively were considered to be significant. Data are representative of 2–3 independent experiments.

    Article Snippet: Anti-hexokinase II (2106), anti-BAX (2772), anti-Bcl-x L (2762), anti-caspase 3 (9662), anti-caspase 9 (9502), anti-BID (2002), anti-GAPDH (2118), and anti-caspase 8 (Asp387, 14071, for flowcytometry) were from Cell Signaling Technology.

    Techniques: Infection, Isolation, Immunoprecipitation, Western Blot, Translocation Assay, Fluorescence, Microscopy, Staining, Expressing

    ( A ) Bcl-x L is required for pro-caspase 8 and RIPK3 accumulation on the mitochondria of H37Rv infected Mφ. Human Mφ were transfected with Bcl-x L or scrambled control (Scr) siRNA and then infected with H37Rv (MOI 10). Equal amounts of purified mitochondria from non-infected and H37Rv-infected Mφ were subjected to Western blot analysis to determine the levels of pro-caspase 8 and RIPK3. ( B ) Caspase inhibition of H37Ra infected Mφ enables RIPK3/Bcl-x L accumulation on mitochondria. After treatment with the caspase inhibitor z-IETD pro-caspase 8, RIPK3 and Bcl-x L accumulates on the mitochondria of H37Ra infected Mφ. Mitochondria were isolated from H37Rv or H37Ra-infected Mφ treated with or without z-IETD (10 μmol) and subjected to Western blot analysis using anti caspase 8, anti-RIPK3 and anti-Bcl-x L ab. Equal amounts of the proteins were subjected to Western blot analysis for the evaluation of RIPK3 and Bcl-x L levels. ( C ) Mφ treated with Bcl-x L or scrambled control siRNA (Scr) were infected with H37Rv (MOI 10) and cell death was assessed after 48 h using Live/Dead fixable dead cell stain kits (Invitrogen). (D) z-IETD blocks activation of the apoptotic caspase 9 and 3 essential for apoptosis induction. Equal amounts of cell lysates of Mφ infected with H37Ra (pro-apoptotic strain) treated with or without z-IETD (10 μmol) were subjected to Western blot analysis for evaluating active caspase 3 and 9. ( E ) RIPK3 is required for accumulation of mitochondrial pro-caspase 8 and Bcl-x L . Mitochondria and cytosolic fraction of H37Rv-infected Mφ treated with RIPK3 or Scr siRNA were subjected to Western blot analysis and the levels of RIPK3, pro-caspase 8 and Bcl-x L were evaluated. (F ) Silencing of the RIPK3 gene activates apoptosis executor caspase 3 in H37Rv infected Mφ. Mφ treated with RIPK3 or Scr siRNA were infected with H37Rv. Cell lysate was collected and equal amounts subjected to Western blot analysis for cleaved caspase 3. ( G ) Bid processing in H37Ra and H37Rv-infected Mφ. Mitochondria were isolated from H37Ra (right panel) or H37Rv (left panel) -infected Mφ and the kinetics of BID processing and tBID accumulation were assessed by Western blotting. ( H ) Silencing of the BAX gene in H37Ra-infected Mφ blocks caspase 3 activation, a marker for apoptosis. Mφ treated with BAX or Scr siRNA were infected with H37Ra or H37Rv (MOI 10). Cell lysate was collected after 0 and 24 h and equal amounts subjected to Western blot analysis for pro-caspase 3 and cleaved caspase 3. VDAC and GAPDH were used as a loading control. Results are expressed as mean ± SD. Data were analyzed using one-way ANOVA. *, Values of P < 0.05 were considered to be significant. Data are representative of four independent experiments.

    Journal: Mucosal immunology

    Article Title: Bcl-x L mediates RIPK3-dependent necrosis in M. tuberculosis -infected macrophages

    doi: 10.1038/mi.2017.12

    Figure Lengend Snippet: ( A ) Bcl-x L is required for pro-caspase 8 and RIPK3 accumulation on the mitochondria of H37Rv infected Mφ. Human Mφ were transfected with Bcl-x L or scrambled control (Scr) siRNA and then infected with H37Rv (MOI 10). Equal amounts of purified mitochondria from non-infected and H37Rv-infected Mφ were subjected to Western blot analysis to determine the levels of pro-caspase 8 and RIPK3. ( B ) Caspase inhibition of H37Ra infected Mφ enables RIPK3/Bcl-x L accumulation on mitochondria. After treatment with the caspase inhibitor z-IETD pro-caspase 8, RIPK3 and Bcl-x L accumulates on the mitochondria of H37Ra infected Mφ. Mitochondria were isolated from H37Rv or H37Ra-infected Mφ treated with or without z-IETD (10 μmol) and subjected to Western blot analysis using anti caspase 8, anti-RIPK3 and anti-Bcl-x L ab. Equal amounts of the proteins were subjected to Western blot analysis for the evaluation of RIPK3 and Bcl-x L levels. ( C ) Mφ treated with Bcl-x L or scrambled control siRNA (Scr) were infected with H37Rv (MOI 10) and cell death was assessed after 48 h using Live/Dead fixable dead cell stain kits (Invitrogen). (D) z-IETD blocks activation of the apoptotic caspase 9 and 3 essential for apoptosis induction. Equal amounts of cell lysates of Mφ infected with H37Ra (pro-apoptotic strain) treated with or without z-IETD (10 μmol) were subjected to Western blot analysis for evaluating active caspase 3 and 9. ( E ) RIPK3 is required for accumulation of mitochondrial pro-caspase 8 and Bcl-x L . Mitochondria and cytosolic fraction of H37Rv-infected Mφ treated with RIPK3 or Scr siRNA were subjected to Western blot analysis and the levels of RIPK3, pro-caspase 8 and Bcl-x L were evaluated. (F ) Silencing of the RIPK3 gene activates apoptosis executor caspase 3 in H37Rv infected Mφ. Mφ treated with RIPK3 or Scr siRNA were infected with H37Rv. Cell lysate was collected and equal amounts subjected to Western blot analysis for cleaved caspase 3. ( G ) Bid processing in H37Ra and H37Rv-infected Mφ. Mitochondria were isolated from H37Ra (right panel) or H37Rv (left panel) -infected Mφ and the kinetics of BID processing and tBID accumulation were assessed by Western blotting. ( H ) Silencing of the BAX gene in H37Ra-infected Mφ blocks caspase 3 activation, a marker for apoptosis. Mφ treated with BAX or Scr siRNA were infected with H37Ra or H37Rv (MOI 10). Cell lysate was collected after 0 and 24 h and equal amounts subjected to Western blot analysis for pro-caspase 3 and cleaved caspase 3. VDAC and GAPDH were used as a loading control. Results are expressed as mean ± SD. Data were analyzed using one-way ANOVA. *, Values of P < 0.05 were considered to be significant. Data are representative of four independent experiments.

    Article Snippet: Anti-hexokinase II (2106), anti-BAX (2772), anti-Bcl-x L (2762), anti-caspase 3 (9662), anti-caspase 9 (9502), anti-BID (2002), anti-GAPDH (2118), and anti-caspase 8 (Asp387, 14071, for flowcytometry) were from Cell Signaling Technology.

    Techniques: Infection, Transfection, Purification, Western Blot, Inhibition, Isolation, Staining, Activation Assay, Marker

    ( A ) RIPK3 is required for MPT in H37Rv infected Mφ. Mφ were transfected with RIPK3 or scrambled (Scr) control siRNA and were then infected with H37Rv (MOI 10). Cationic dye (DiOC6 (3) ) release from mitochondria (a measure for MPT) was measured at 48 and 72 h after infection. ( B and C ) Inhibition of CypD reduces ROS-dependent necrosis. ( B ) Equal numbers of CsA-treated (5 μM) and untreated Mφ were infected with H37Rv (MOI 5 or 10). After 48 h of infection ROS accumulation ( B ) was measured by FACS analysis using the fluorescent dye CM-H 2 XRos and cell viability ( C ) was determined using the Live-Dead Assay. ( D ) CypD inactivation reduces translocation of RIPK3, pro-caspase 8, Bcl-x L and HKII to the mitochondria in H37Rv-infected Mφ at 24 h. Equal amounts of mitochondria from H37Rv-infected Mφ treated with or without CsA (5 μM) were subjected to Western blot analysis for RIPK3, pro-caspase 8, Bcl-x L and HKII after 24 h of infection. ( E ) Mitochondria from H37Ra or H37Rv infected Mφ were subjected to IP with anti-ANT ab and were then subjected to Western blot analysis for CypD. ( F ) CypD - ANT interaction is augmented in H37Rv-infected Mφ and is inhibited by inactivation of CypD with CsA (5 μM). ( G ) Top panel: RIPK3 is required for CypD - ANT interaction on the mitochondria. After 24h of infection, mitochondria from H37Rv-infected Mφ transfected with RIPK3 or scrambled (Scr) control siRNA were subjected to IP with anti-ANT ab and were then analyzed by Western blot for CypD. ANT was used as a loading control. Bottom panel: Silencing efficiency of RIPK3 siRNA. VDAC was used as a loading control. Results are expressed as mean ± SE, using nonparametric Student t test. *, Values of P < 0.05 were considered to be significant. Data are representative of three independent experiments.

    Journal: Mucosal immunology

    Article Title: Bcl-x L mediates RIPK3-dependent necrosis in M. tuberculosis -infected macrophages

    doi: 10.1038/mi.2017.12

    Figure Lengend Snippet: ( A ) RIPK3 is required for MPT in H37Rv infected Mφ. Mφ were transfected with RIPK3 or scrambled (Scr) control siRNA and were then infected with H37Rv (MOI 10). Cationic dye (DiOC6 (3) ) release from mitochondria (a measure for MPT) was measured at 48 and 72 h after infection. ( B and C ) Inhibition of CypD reduces ROS-dependent necrosis. ( B ) Equal numbers of CsA-treated (5 μM) and untreated Mφ were infected with H37Rv (MOI 5 or 10). After 48 h of infection ROS accumulation ( B ) was measured by FACS analysis using the fluorescent dye CM-H 2 XRos and cell viability ( C ) was determined using the Live-Dead Assay. ( D ) CypD inactivation reduces translocation of RIPK3, pro-caspase 8, Bcl-x L and HKII to the mitochondria in H37Rv-infected Mφ at 24 h. Equal amounts of mitochondria from H37Rv-infected Mφ treated with or without CsA (5 μM) were subjected to Western blot analysis for RIPK3, pro-caspase 8, Bcl-x L and HKII after 24 h of infection. ( E ) Mitochondria from H37Ra or H37Rv infected Mφ were subjected to IP with anti-ANT ab and were then subjected to Western blot analysis for CypD. ( F ) CypD - ANT interaction is augmented in H37Rv-infected Mφ and is inhibited by inactivation of CypD with CsA (5 μM). ( G ) Top panel: RIPK3 is required for CypD - ANT interaction on the mitochondria. After 24h of infection, mitochondria from H37Rv-infected Mφ transfected with RIPK3 or scrambled (Scr) control siRNA were subjected to IP with anti-ANT ab and were then analyzed by Western blot for CypD. ANT was used as a loading control. Bottom panel: Silencing efficiency of RIPK3 siRNA. VDAC was used as a loading control. Results are expressed as mean ± SE, using nonparametric Student t test. *, Values of P < 0.05 were considered to be significant. Data are representative of three independent experiments.

    Article Snippet: Anti-hexokinase II (2106), anti-BAX (2772), anti-Bcl-x L (2762), anti-caspase 3 (9662), anti-caspase 9 (9502), anti-BID (2002), anti-GAPDH (2118), and anti-caspase 8 (Asp387, 14071, for flowcytometry) were from Cell Signaling Technology.

    Techniques: Infection, Transfection, Inhibition, Live Dead Assay, Translocation Assay, Western Blot

    In Mφ infected with virulent Mtb RIPK3 and pro-caspase 8 present in the cytosol translocate to the mitochondria in presence of Bcl-x L and RIPK3 is activated RIPK3 enhances binding of HKII to VDAC on the outer mitochondrial membrane controlling mitochondrial glycolysis. At the same time activated RIPK3 triggers CypD-dependent formation of the mitochondrial permeability transition (MPT) pore via interaction between ANT and VDAC leading to leakage of the electron chain. Both mechanisms seem to be required for increasing ROS-dependent necrosis (right). In Mφ infected with avirulent Mtb the RIPK3 and caspase 8 also translocate to the mitochondria but this step is quickly followed by activation of caspase 8 and degradation of RIPK3. Oligomerization of BAX and BAK, which in turn allows the release of pro-apoptotic molecules (e.g. cytochrome c) leads to apoptosis (left). The exact action mechanism of Bcl-x L function is unknown.

    Journal: Mucosal immunology

    Article Title: Bcl-x L mediates RIPK3-dependent necrosis in M. tuberculosis -infected macrophages

    doi: 10.1038/mi.2017.12

    Figure Lengend Snippet: In Mφ infected with virulent Mtb RIPK3 and pro-caspase 8 present in the cytosol translocate to the mitochondria in presence of Bcl-x L and RIPK3 is activated RIPK3 enhances binding of HKII to VDAC on the outer mitochondrial membrane controlling mitochondrial glycolysis. At the same time activated RIPK3 triggers CypD-dependent formation of the mitochondrial permeability transition (MPT) pore via interaction between ANT and VDAC leading to leakage of the electron chain. Both mechanisms seem to be required for increasing ROS-dependent necrosis (right). In Mφ infected with avirulent Mtb the RIPK3 and caspase 8 also translocate to the mitochondria but this step is quickly followed by activation of caspase 8 and degradation of RIPK3. Oligomerization of BAX and BAK, which in turn allows the release of pro-apoptotic molecules (e.g. cytochrome c) leads to apoptosis (left). The exact action mechanism of Bcl-x L function is unknown.

    Article Snippet: Anti-hexokinase II (2106), anti-BAX (2772), anti-Bcl-x L (2762), anti-caspase 3 (9662), anti-caspase 9 (9502), anti-BID (2002), anti-GAPDH (2118), and anti-caspase 8 (Asp387, 14071, for flowcytometry) were from Cell Signaling Technology.

    Techniques: Infection, Binding Assay, Permeability, Activation Assay