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  • 86
    Becton Dickinson apoptosis
    MPA but not NET-A or P4 increases Vpr-mediated <t>apoptosis</t> in a dose-dependent manner. (A) PBMCs were treated with or without 5 µM Vpr peptide (amino acids 52–96) and increasing concentrations of MPA, NET-A or P4 for 24 hrs. The graph shows results pooled from two independent experiments with samples from three donors. (B) PBMCs were treated with 100 nM MPA, 10 µM NET-A, 1 µM P4 or in combination with 5 µM Vpr peptide (amino acids 52–96) for 24 hrs. Cells were stained and acquired by flow cytometry as described in the materials and methods . A tryptic BSA digest served as a control wherever Vpr peptide was not added, as for results in figure 4 . The histogram shows results pooled from two independent experiments with samples from three different donors compared to those in figure A. donors. Statistical trend analysis for panel A was performed by the Wilcox rank-sum test, as further extended by Cuzick [85] , and showed a significant trend only for MPA plus Vpr (p = 0.012) and P4 minus Vpr (p = 0.012). Statistical significance in panel B was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p
    Apoptosis, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Roche apoptosis
    Activation of gp120-primed DC reduced the expression of Bcl2 and activated Akt, and the induction of cell <t>apoptosis</t> is ASK1-dependent. ( A ) moDC treated with immune-complex gp120 ADA were exposed to CL40L Tf or mock Tf, or LPS, TNFα, IL-1β for 3 d, and cellular proteins of moDC (recovered from coculture) were extracted for Western blotting analysis. MoDC treated by anti-His Ab (used to cross-link recombinant gp120 ADA ) were used as a control (control DC). *p-ASK1 represent results from untreated moDC (lane 1) and DC treated as indicated but no gp120 pulsing (lanes 2–6), whereas #p-ASK-1 represents results from pre-treatment of anti-DC-SIGN mAbs. Data are representative of 3 experiments. ( B ) moDC were transfected with siRNA against human ASK1 or control siRNA (scrambled) before pulse with immune-complex gp120 and subsequent exposure to CD40L Tf, LPS, TNFα or IL-1β. Data are expressed as mean ± SD from 3 experiments. **p
    Apoptosis, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    99
    Millipore apoptosis
    Withanolides induce cellular stress response in NB cells IMR 32 cells were preloaded with ROS indicator CM-H 2 DCFDA prior to withanolides exposure and alteration in oxidation potential was measured. ( A and B ) Fluorescent intensity measurement revelaed the induction of ROS after 4 h of treatment in a dose dependent manner and complete blocking of this effect by 5 mM NAC co-treatment. Immunoblot analysis of the stress response, heat shock proteins demonstrated increased expression levels of HSP32 and HSP70, and decreased expression of HSF1 after 24 h of treatment with increasing concentrations of withanolides. When the cells were pre-treated with NAC, cleavage of PARP that is an indicator of <t>apoptosis</t> was blocked and the increase in the expression levels of the heat shock proteins HSP32 and HSP70 as well as decrease in expression levels of mTOR pathway proteins and Iκ-Bα are blocked after NAC treatment, indicating attenuation of the anti-proliferative properties of withanolides by NAC ( C – E ).
    Apoptosis, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore situ apoptosis detection kit
    Concomitant loss of MLKL and Caspase-8, but not loss of RIPK1 kinase activity or combined loss of RIPK3 and Caspase-8, promotes survival of LUBAC-deficient mice a, Representative images of E10.5 ( n =6 embryos/genotype), scale bar: 2 mm, E14.5 ( n =12 Ripk1 K45A Hoil-1 +/- , n =5 Ripk1 K45A Hoil-1 -/- embryos/genotype) and E15.5 embryos ( n =3 embryos/genotype). Scale bar: 5 mm. *: poor yolk sac vascularisation. b, f, Representative images of yolk sac vascularisation (PECAM-1, red) and <t>apoptosis</t> (cleaved (cl.) Caspase-3, green) at E14.5 (b) or E13.5 (f) and quantification. Mean ± s.e.m. and P values from unpaired two-tailed t -tests (b) or one-way ANOVA (f) are shown. Scale bar: 50 µm. c, FADD-IP in MEFs treated for 3 h with zVAD-fmk ± TNF ( n . d, Cell death by PI incorporation in MEFs ± TNF (10 ng/ml) or LT-α. Mean ± s.e.m. ( n =3 independent experiments) and P values (**** P
    Situ Apoptosis Detection Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Apoptosis activator 2 promotes the cytochrome c dependent oligomerization of Apaf 1 into the mature apoptosome thus increasing procaspase 9 processing and subsequent caspase 3 activation This compound has been
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    Apoptosis Kit for detection of apoptosis in the research laboratory
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    Image Search Results


    MPA but not NET-A or P4 increases Vpr-mediated apoptosis in a dose-dependent manner. (A) PBMCs were treated with or without 5 µM Vpr peptide (amino acids 52–96) and increasing concentrations of MPA, NET-A or P4 for 24 hrs. The graph shows results pooled from two independent experiments with samples from three donors. (B) PBMCs were treated with 100 nM MPA, 10 µM NET-A, 1 µM P4 or in combination with 5 µM Vpr peptide (amino acids 52–96) for 24 hrs. Cells were stained and acquired by flow cytometry as described in the materials and methods . A tryptic BSA digest served as a control wherever Vpr peptide was not added, as for results in figure 4 . The histogram shows results pooled from two independent experiments with samples from three different donors compared to those in figure A. donors. Statistical trend analysis for panel A was performed by the Wilcox rank-sum test, as further extended by Cuzick [85] , and showed a significant trend only for MPA plus Vpr (p = 0.012) and P4 minus Vpr (p = 0.012). Statistical significance in panel B was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: MPA but not NET-A or P4 increases Vpr-mediated apoptosis in a dose-dependent manner. (A) PBMCs were treated with or without 5 µM Vpr peptide (amino acids 52–96) and increasing concentrations of MPA, NET-A or P4 for 24 hrs. The graph shows results pooled from two independent experiments with samples from three donors. (B) PBMCs were treated with 100 nM MPA, 10 µM NET-A, 1 µM P4 or in combination with 5 µM Vpr peptide (amino acids 52–96) for 24 hrs. Cells were stained and acquired by flow cytometry as described in the materials and methods . A tryptic BSA digest served as a control wherever Vpr peptide was not added, as for results in figure 4 . The histogram shows results pooled from two independent experiments with samples from three different donors compared to those in figure A. donors. Statistical trend analysis for panel A was performed by the Wilcox rank-sum test, as further extended by Cuzick [85] , and showed a significant trend only for MPA plus Vpr (p = 0.012) and P4 minus Vpr (p = 0.012). Statistical significance in panel B was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Staining, Flow Cytometry, Cytometry

    HIV-1-mediated apoptosis is enhanced in the presence of Dex and MPA. PBMCs were activated in the presence of PHA and rhIL-2 for 3 days at 37°C as described. Pseudotyped HIV-1 virus or control medium without virus was added to the cells, followed by incubation for a further 3 days to allow infection. Cells were then treated with vehicle (EtOH) or 100 nM Dex or MPA for an additional 24 hrs. Acquisition and analysis was carried out as described in the methods . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *, **, and *** indicate p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: HIV-1-mediated apoptosis is enhanced in the presence of Dex and MPA. PBMCs were activated in the presence of PHA and rhIL-2 for 3 days at 37°C as described. Pseudotyped HIV-1 virus or control medium without virus was added to the cells, followed by incubation for a further 3 days to allow infection. Cells were then treated with vehicle (EtOH) or 100 nM Dex or MPA for an additional 24 hrs. Acquisition and analysis was carried out as described in the methods . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *, **, and *** indicate p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Incubation, Infection

    The GR is involved in GC- and Vpr-mediated apoptosis in CD4 + T-cells. PBMCs were treated with 100 nM Dex or 1 µM RU486 in the absence or presence of 5 µM Vpr peptide (amino acids 52–96) for 24 hrs. A tryptic BSA digest added at an equivalent mass/volume ratio of peptide, served as a control wherever Vpr peptide was present. Cells were obtained and stained as described in the methods . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: The GR is involved in GC- and Vpr-mediated apoptosis in CD4 + T-cells. PBMCs were treated with 100 nM Dex or 1 µM RU486 in the absence or presence of 5 µM Vpr peptide (amino acids 52–96) for 24 hrs. A tryptic BSA digest added at an equivalent mass/volume ratio of peptide, served as a control wherever Vpr peptide was present. Cells were obtained and stained as described in the methods . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Staining

    The GR is involved in MPA- and Vpr-mediated apoptosis in CD4 + T-cells. PBMCs were treated with vehicle (EtOH), 100 nM MPA, 1 µM RU486 or 100 nM MPA plus 1 µM RU486, in the absence (A) or presence (B) of 5 µM Vpr peptide for 24 hrs. Cells were stained and acquired by flow cytometry as indicated in the methods . In A cells were not incubated with balanced isotonic glucose-HEPES buffer while in B, this buffer was used and a tryptic BSA digest served as a control wherever Vpr peptide was not added, as described in Methods . The histograms show pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: The GR is involved in MPA- and Vpr-mediated apoptosis in CD4 + T-cells. PBMCs were treated with vehicle (EtOH), 100 nM MPA, 1 µM RU486 or 100 nM MPA plus 1 µM RU486, in the absence (A) or presence (B) of 5 µM Vpr peptide for 24 hrs. Cells were stained and acquired by flow cytometry as indicated in the methods . In A cells were not incubated with balanced isotonic glucose-HEPES buffer while in B, this buffer was used and a tryptic BSA digest served as a control wherever Vpr peptide was not added, as described in Methods . The histograms show pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Staining, Flow Cytometry, Cytometry, Incubation

    Apoptosis induction by Dex and MPA is most likely mediated primarily through the GR. (A) PBMCs were treated with vehicle (EtOH), 100 nM MPA, 10 nM Ald, 100 nM E2, 100 nM Mib, 100 nM R5020, 10 µM NET-A or 10 µM NET for 24 hrs at 37°C. Cells were surface stained with ant-CD3 and anti-CD4 antibodies, and apoptosis was detected using flow cytometry as described in Figure 1 . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *** indicates p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: Apoptosis induction by Dex and MPA is most likely mediated primarily through the GR. (A) PBMCs were treated with vehicle (EtOH), 100 nM MPA, 10 nM Ald, 100 nM E2, 100 nM Mib, 100 nM R5020, 10 µM NET-A or 10 µM NET for 24 hrs at 37°C. Cells were surface stained with ant-CD3 and anti-CD4 antibodies, and apoptosis was detected using flow cytometry as described in Figure 1 . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *** indicates p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Staining, Flow Cytometry, Cytometry

    GC and Vpr differentially regulate key genes involved in apoptosis. PBMCs were treated with or without 5 µM Vpr peptide as described previously and treated with or without 100 nM Dex, MPA, NET-A or P4 for 24 hrs. After treatment, RNA was extracted, reverse transcribed, and Bcl-2 (A) or, Bim (B) mRNA expression was measured by real time PCR, normalising to GAPDH expression levels. The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *, **, and *** indicate p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: GC and Vpr differentially regulate key genes involved in apoptosis. PBMCs were treated with or without 5 µM Vpr peptide as described previously and treated with or without 100 nM Dex, MPA, NET-A or P4 for 24 hrs. After treatment, RNA was extracted, reverse transcribed, and Bcl-2 (A) or, Bim (B) mRNA expression was measured by real time PCR, normalising to GAPDH expression levels. The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *, **, and *** indicate p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    The GR is involved in Vpr-mediated apoptosis in CD4 + T-cells. PBMCs were treated with 1 µM RU486 in the absence or presence of 5 µM Vpr peptide (amino acids 52–96) for 24 hrs. A tryptic BSA digest served as a control (bars 1 and 2) wherever Vpr peptide was not added and was added at an equivalent mass/volume of peptide. Cells were obtained and stained as described in the methods . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: The GR is involved in Vpr-mediated apoptosis in CD4 + T-cells. PBMCs were treated with 1 µM RU486 in the absence or presence of 5 µM Vpr peptide (amino acids 52–96) for 24 hrs. A tryptic BSA digest served as a control (bars 1 and 2) wherever Vpr peptide was not added and was added at an equivalent mass/volume of peptide. Cells were obtained and stained as described in the methods . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Staining

    Dose-dependent apoptosis induction with Dex, MPA, NET-A and P4 in CD4 + T-cells. PBMCs were treated with vehicle (EtOH), Dex, MPA, NET-A or P4 at the concentrations indicated for 24 hrs at 37°C. Cells were stained and analysed as described for Figure 1 . The figure shows pooled results from two independent experiments with samples from three donors. Error bars represent standard deviation. Statistical trend analysis for each dose response was performed by the Wilcox rank-sum test, as further extended by Cuzick [85] , and showed a significant trend only for Dex (p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: Dose-dependent apoptosis induction with Dex, MPA, NET-A and P4 in CD4 + T-cells. PBMCs were treated with vehicle (EtOH), Dex, MPA, NET-A or P4 at the concentrations indicated for 24 hrs at 37°C. Cells were stained and analysed as described for Figure 1 . The figure shows pooled results from two independent experiments with samples from three donors. Error bars represent standard deviation. Statistical trend analysis for each dose response was performed by the Wilcox rank-sum test, as further extended by Cuzick [85] , and showed a significant trend only for Dex (p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Staining, Standard Deviation

    The progestin MPA, but not NET-A or P4, induces apoptosis in CD4 + T-cells. Cells were treated with or without 100 nM Dex, 100 nM F, 1 µM MPA, 10 µM NET-A, 1 µM P4 or vehicle control (EtOH) for 24 hrs. Cells were stained with anti-CD4, anti-CD14, annexin V and 7-AAD using the Apoptosis Detection kit I (BD biosciences). (A) Gating strategy and representative zebra plots of untreated (EtOH), MPA or Dex treated PBMCs. (B) The histogram shows pooled results from two independent experiments with samples from three donors. Data were acquired on a FACS calibur system (BD Biosciences) and analyzed using Flo-Jo software (Tree Star Inc., San Carlos, CA, USA). Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *, **, and *** indicate p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: The progestin MPA, but not NET-A or P4, induces apoptosis in CD4 + T-cells. Cells were treated with or without 100 nM Dex, 100 nM F, 1 µM MPA, 10 µM NET-A, 1 µM P4 or vehicle control (EtOH) for 24 hrs. Cells were stained with anti-CD4, anti-CD14, annexin V and 7-AAD using the Apoptosis Detection kit I (BD biosciences). (A) Gating strategy and representative zebra plots of untreated (EtOH), MPA or Dex treated PBMCs. (B) The histogram shows pooled results from two independent experiments with samples from three donors. Data were acquired on a FACS calibur system (BD Biosciences) and analyzed using Flo-Jo software (Tree Star Inc., San Carlos, CA, USA). Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *, **, and *** indicate p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Staining, FACS, Software

    Activation of gp120-primed DC reduced the expression of Bcl2 and activated Akt, and the induction of cell apoptosis is ASK1-dependent. ( A ) moDC treated with immune-complex gp120 ADA were exposed to CL40L Tf or mock Tf, or LPS, TNFα, IL-1β for 3 d, and cellular proteins of moDC (recovered from coculture) were extracted for Western blotting analysis. MoDC treated by anti-His Ab (used to cross-link recombinant gp120 ADA ) were used as a control (control DC). *p-ASK1 represent results from untreated moDC (lane 1) and DC treated as indicated but no gp120 pulsing (lanes 2–6), whereas #p-ASK-1 represents results from pre-treatment of anti-DC-SIGN mAbs. Data are representative of 3 experiments. ( B ) moDC were transfected with siRNA against human ASK1 or control siRNA (scrambled) before pulse with immune-complex gp120 and subsequent exposure to CD40L Tf, LPS, TNFα or IL-1β. Data are expressed as mean ± SD from 3 experiments. **p

    Journal: PLoS Pathogens

    Article Title: Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

    doi: 10.1371/journal.ppat.1003100

    Figure Lengend Snippet: Activation of gp120-primed DC reduced the expression of Bcl2 and activated Akt, and the induction of cell apoptosis is ASK1-dependent. ( A ) moDC treated with immune-complex gp120 ADA were exposed to CL40L Tf or mock Tf, or LPS, TNFα, IL-1β for 3 d, and cellular proteins of moDC (recovered from coculture) were extracted for Western blotting analysis. MoDC treated by anti-His Ab (used to cross-link recombinant gp120 ADA ) were used as a control (control DC). *p-ASK1 represent results from untreated moDC (lane 1) and DC treated as indicated but no gp120 pulsing (lanes 2–6), whereas #p-ASK-1 represents results from pre-treatment of anti-DC-SIGN mAbs. Data are representative of 3 experiments. ( B ) moDC were transfected with siRNA against human ASK1 or control siRNA (scrambled) before pulse with immune-complex gp120 and subsequent exposure to CD40L Tf, LPS, TNFα or IL-1β. Data are expressed as mean ± SD from 3 experiments. **p

    Article Snippet: Alternatively, for biosafety reasons after co-culture with activated CD4 T cells, apoptosis was determined by terminal deoxynucleotidyl transferase (TdT) dUTP nick end labelling (TUNEL) assay using the In-Situ Cell Death Detection Kit (Roche Diagnostics, Inc.), according to manufacturer's instructions; the recovered moDC were fixed, permeabilized and subjected to TUNEL reaction mix incubation at 37°C for 1 h, followed by cytometric analysis.

    Techniques: Activation Assay, Expressing, Western Blot, Recombinant, Transfection

    Cross-linked gp120 sensitizes DC through DC-SIGN and MCLRs for CD40L-mediated apoptosis. ( A , B ) moDC were pretreated with anti-DC-SIGN mAbs or isotype control Ab before pulse with cross-linked gp120 ADA and co-culture for 3 d with autologous activated CD4 T cells, and subsequently subjected to cell viability assay. Data are representative of 3 experiments and are expressed as mean ± SD from 3 experiments in B . ( C, D ) moDC were treated with cross-linked recombinant gp120 ADA with or without pre-treatment by soluble ICAM-3-Fc chimeric protein, anti-CD4 plus anti-CCR5 mAbs, or anti-DC-SIGN mAbs. Cells were subsequently co-cultured with mock- or CD40L-transfected (CD40L Tf) cells for 3 d. Data are representative of 7 experiments in panel C and are expressed as mean ± SD (n = 7) in D ; ***p

    Journal: PLoS Pathogens

    Article Title: Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

    doi: 10.1371/journal.ppat.1003100

    Figure Lengend Snippet: Cross-linked gp120 sensitizes DC through DC-SIGN and MCLRs for CD40L-mediated apoptosis. ( A , B ) moDC were pretreated with anti-DC-SIGN mAbs or isotype control Ab before pulse with cross-linked gp120 ADA and co-culture for 3 d with autologous activated CD4 T cells, and subsequently subjected to cell viability assay. Data are representative of 3 experiments and are expressed as mean ± SD from 3 experiments in B . ( C, D ) moDC were treated with cross-linked recombinant gp120 ADA with or without pre-treatment by soluble ICAM-3-Fc chimeric protein, anti-CD4 plus anti-CCR5 mAbs, or anti-DC-SIGN mAbs. Cells were subsequently co-cultured with mock- or CD40L-transfected (CD40L Tf) cells for 3 d. Data are representative of 7 experiments in panel C and are expressed as mean ± SD (n = 7) in D ; ***p

    Article Snippet: Alternatively, for biosafety reasons after co-culture with activated CD4 T cells, apoptosis was determined by terminal deoxynucleotidyl transferase (TdT) dUTP nick end labelling (TUNEL) assay using the In-Situ Cell Death Detection Kit (Roche Diagnostics, Inc.), according to manufacturer's instructions; the recovered moDC were fixed, permeabilized and subjected to TUNEL reaction mix incubation at 37°C for 1 h, followed by cytometric analysis.

    Techniques: Co-Culture Assay, Viability Assay, Recombinant, Cell Culture, Transfection

    Sera from HIV-1(+) individuals can sensitize moDC for DC-SIGN dependent CD40L-mediated apoptosis. ( A ) moDC were treated with HIV(+) serum before or after immunoprecipitation (IP) with anti-gp120 mAbs, or with or without anti-DC-SIGN or isotype control mAbs, and subsequently co-cultured with autologous activated CD4 T cells. After 3 d, cells were harvested and subjected to TUNEL assays. Cell death was assessed as the percentage of cells expressing terminal deoxynucleotidyl transferase (TdT). DC pulsed with HIV(+) serum without coculture with activated CD4 T cells (top panel) were also used as a control. Data are representative of 4 experiments. ( B ) MoDCs were treated with anti-DC-SIGN mAbs, isotype control Ab, or anti-CD40L mAb before pulse with HIV serum (before or after immunoprecipitation of gp120) and cocultured with activated CD4 T cells. Data are expressed as mean ± SD (n = 4); *p

    Journal: PLoS Pathogens

    Article Title: Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

    doi: 10.1371/journal.ppat.1003100

    Figure Lengend Snippet: Sera from HIV-1(+) individuals can sensitize moDC for DC-SIGN dependent CD40L-mediated apoptosis. ( A ) moDC were treated with HIV(+) serum before or after immunoprecipitation (IP) with anti-gp120 mAbs, or with or without anti-DC-SIGN or isotype control mAbs, and subsequently co-cultured with autologous activated CD4 T cells. After 3 d, cells were harvested and subjected to TUNEL assays. Cell death was assessed as the percentage of cells expressing terminal deoxynucleotidyl transferase (TdT). DC pulsed with HIV(+) serum without coculture with activated CD4 T cells (top panel) were also used as a control. Data are representative of 4 experiments. ( B ) MoDCs were treated with anti-DC-SIGN mAbs, isotype control Ab, or anti-CD40L mAb before pulse with HIV serum (before or after immunoprecipitation of gp120) and cocultured with activated CD4 T cells. Data are expressed as mean ± SD (n = 4); *p

    Article Snippet: Alternatively, for biosafety reasons after co-culture with activated CD4 T cells, apoptosis was determined by terminal deoxynucleotidyl transferase (TdT) dUTP nick end labelling (TUNEL) assay using the In-Situ Cell Death Detection Kit (Roche Diagnostics, Inc.), according to manufacturer's instructions; the recovered moDC were fixed, permeabilized and subjected to TUNEL reaction mix incubation at 37°C for 1 h, followed by cytometric analysis.

    Techniques: Immunoprecipitation, Cell Culture, TUNEL Assay, Expressing

    Cross-linked recombinant gp120 sensitizes moDC for CD40L-mediated apoptosis after co-culture with activated CD4 T cells. ( A ) moDC were treated for 24 h with anti-His mAb alone (control DC, left panels) or with 25 nM gp120 ADA cross-linked with anti-His mAb (gp120-DC, right panels), and co-cultured with autologous activated (upper panels) or naïve (lower panels) CD4 T cells for 3 d. The moDC ( Fig. S1 ) were analyzed for Annexin V (AV) and propidium iodide (PI) expression to assess the extent of apoptosis, as manifested by the percentage of the AV-positive [AV(+)] cells. Data are representative of 5 experiments. ( B ) Apoptosis of moDC was analyzed after treatment with different concentrations of cross-linked recombinant gp120 ADA or gp120 HXBc2 and co-culture with activated CD4 T cells for 3 d. DC treated with monomeric gp120 (not cross-linked with anti-His or anti-FLAG mAb) were used as a control. Data represent mean ± SD from 5 experiments; **p

    Journal: PLoS Pathogens

    Article Title: Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

    doi: 10.1371/journal.ppat.1003100

    Figure Lengend Snippet: Cross-linked recombinant gp120 sensitizes moDC for CD40L-mediated apoptosis after co-culture with activated CD4 T cells. ( A ) moDC were treated for 24 h with anti-His mAb alone (control DC, left panels) or with 25 nM gp120 ADA cross-linked with anti-His mAb (gp120-DC, right panels), and co-cultured with autologous activated (upper panels) or naïve (lower panels) CD4 T cells for 3 d. The moDC ( Fig. S1 ) were analyzed for Annexin V (AV) and propidium iodide (PI) expression to assess the extent of apoptosis, as manifested by the percentage of the AV-positive [AV(+)] cells. Data are representative of 5 experiments. ( B ) Apoptosis of moDC was analyzed after treatment with different concentrations of cross-linked recombinant gp120 ADA or gp120 HXBc2 and co-culture with activated CD4 T cells for 3 d. DC treated with monomeric gp120 (not cross-linked with anti-His or anti-FLAG mAb) were used as a control. Data represent mean ± SD from 5 experiments; **p

    Article Snippet: Alternatively, for biosafety reasons after co-culture with activated CD4 T cells, apoptosis was determined by terminal deoxynucleotidyl transferase (TdT) dUTP nick end labelling (TUNEL) assay using the In-Situ Cell Death Detection Kit (Roche Diagnostics, Inc.), according to manufacturer's instructions; the recovered moDC were fixed, permeabilized and subjected to TUNEL reaction mix incubation at 37°C for 1 h, followed by cytometric analysis.

    Techniques: Recombinant, Co-Culture Assay, Cell Culture, Expressing

    Cross-linked recombinant gp120 or HIV(+) serum sensitizes moDC for apoptosis after activation by LPS, TNF-α or IL-1β, and DC-SIGN(+) cells in HIV(+) blood are pre-sensitized for LPS/TNFα/IL-1β-induced apoptosis. ( A,B ) moDC were treated with gp120 ADA in the presence of isotype control or anti-DC-SIGN Abs and subsequently cultured in the absence or presence of 100 ng/ml LPS for 3 d. Data are representative of 5 experiments in A and are expressed as mean ± SD in B ; **p

    Journal: PLoS Pathogens

    Article Title: Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

    doi: 10.1371/journal.ppat.1003100

    Figure Lengend Snippet: Cross-linked recombinant gp120 or HIV(+) serum sensitizes moDC for apoptosis after activation by LPS, TNF-α or IL-1β, and DC-SIGN(+) cells in HIV(+) blood are pre-sensitized for LPS/TNFα/IL-1β-induced apoptosis. ( A,B ) moDC were treated with gp120 ADA in the presence of isotype control or anti-DC-SIGN Abs and subsequently cultured in the absence or presence of 100 ng/ml LPS for 3 d. Data are representative of 5 experiments in A and are expressed as mean ± SD in B ; **p

    Article Snippet: Alternatively, for biosafety reasons after co-culture with activated CD4 T cells, apoptosis was determined by terminal deoxynucleotidyl transferase (TdT) dUTP nick end labelling (TUNEL) assay using the In-Situ Cell Death Detection Kit (Roche Diagnostics, Inc.), according to manufacturer's instructions; the recovered moDC were fixed, permeabilized and subjected to TUNEL reaction mix incubation at 37°C for 1 h, followed by cytometric analysis.

    Techniques: Recombinant, Activation Assay, Cell Culture

    CD40L-mediated apoptosis of HIV(+) serum-pulsed DC is proportional to viral loads and predominantly induced by the 100–1000 kDa fraction. ( A ) moDC were treated with HIV-1(+) sera with viral copy numbers > 400,000/ml or

    Journal: PLoS Pathogens

    Article Title: Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

    doi: 10.1371/journal.ppat.1003100

    Figure Lengend Snippet: CD40L-mediated apoptosis of HIV(+) serum-pulsed DC is proportional to viral loads and predominantly induced by the 100–1000 kDa fraction. ( A ) moDC were treated with HIV-1(+) sera with viral copy numbers > 400,000/ml or

    Article Snippet: Alternatively, for biosafety reasons after co-culture with activated CD4 T cells, apoptosis was determined by terminal deoxynucleotidyl transferase (TdT) dUTP nick end labelling (TUNEL) assay using the In-Situ Cell Death Detection Kit (Roche Diagnostics, Inc.), according to manufacturer's instructions; the recovered moDC were fixed, permeabilized and subjected to TUNEL reaction mix incubation at 37°C for 1 h, followed by cytometric analysis.

    Techniques:

    Freshly-isolated DC-SIGN(+) blood DC underwent DC-SIGN-dependent CD40L-mediated apoptosis and DC-SIGN(+) cells from HIV-1-infected individuals are pre-sensitized for CD40L-mediated apoptosis. ( A ) PBMCs from normal HIV(−) individuals were labelled with anti-CD14 plus either isotype control (left panel) or anti-DC-SIGN (right panel) mAbs and analysed by flow cytometry for cell isolation. Data are representative of 4 experiments. ( B ) Purified CD14(+)DC-SIGN(+) cells were treated with anti-His mAb alone (Control) or anti-His cross-linked recombinant gp120 ADA , in the absence or presence of anti-DC-SIGN mAbs, and subsequently co-cultured with CD40L Tf for 3 d. The non-adherent DC were then harvested and subjected to cell viability assay. Data are representative of 4 experiments. ( C, D ) freshly isolated DC-SIGN(+) cells from HIV(+) and HIV(−) blood were cocultured with mock Tf or CD40L Tf for 3 days and subjected to cell viability assay. Data are representative of 4 experiments in C and expressed as mean ± SD in D . ***p

    Journal: PLoS Pathogens

    Article Title: Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

    doi: 10.1371/journal.ppat.1003100

    Figure Lengend Snippet: Freshly-isolated DC-SIGN(+) blood DC underwent DC-SIGN-dependent CD40L-mediated apoptosis and DC-SIGN(+) cells from HIV-1-infected individuals are pre-sensitized for CD40L-mediated apoptosis. ( A ) PBMCs from normal HIV(−) individuals were labelled with anti-CD14 plus either isotype control (left panel) or anti-DC-SIGN (right panel) mAbs and analysed by flow cytometry for cell isolation. Data are representative of 4 experiments. ( B ) Purified CD14(+)DC-SIGN(+) cells were treated with anti-His mAb alone (Control) or anti-His cross-linked recombinant gp120 ADA , in the absence or presence of anti-DC-SIGN mAbs, and subsequently co-cultured with CD40L Tf for 3 d. The non-adherent DC were then harvested and subjected to cell viability assay. Data are representative of 4 experiments. ( C, D ) freshly isolated DC-SIGN(+) cells from HIV(+) and HIV(−) blood were cocultured with mock Tf or CD40L Tf for 3 days and subjected to cell viability assay. Data are representative of 4 experiments in C and expressed as mean ± SD in D . ***p

    Article Snippet: Alternatively, for biosafety reasons after co-culture with activated CD4 T cells, apoptosis was determined by terminal deoxynucleotidyl transferase (TdT) dUTP nick end labelling (TUNEL) assay using the In-Situ Cell Death Detection Kit (Roche Diagnostics, Inc.), according to manufacturer's instructions; the recovered moDC were fixed, permeabilized and subjected to TUNEL reaction mix incubation at 37°C for 1 h, followed by cytometric analysis.

    Techniques: Isolation, Infection, Flow Cytometry, Cytometry, Cell Isolation, Purification, Recombinant, Cell Culture, Viability Assay

    Withanolides induce cellular stress response in NB cells IMR 32 cells were preloaded with ROS indicator CM-H 2 DCFDA prior to withanolides exposure and alteration in oxidation potential was measured. ( A and B ) Fluorescent intensity measurement revelaed the induction of ROS after 4 h of treatment in a dose dependent manner and complete blocking of this effect by 5 mM NAC co-treatment. Immunoblot analysis of the stress response, heat shock proteins demonstrated increased expression levels of HSP32 and HSP70, and decreased expression of HSF1 after 24 h of treatment with increasing concentrations of withanolides. When the cells were pre-treated with NAC, cleavage of PARP that is an indicator of apoptosis was blocked and the increase in the expression levels of the heat shock proteins HSP32 and HSP70 as well as decrease in expression levels of mTOR pathway proteins and Iκ-Bα are blocked after NAC treatment, indicating attenuation of the anti-proliferative properties of withanolides by NAC ( C – E ).

    Journal: Oncotarget

    Article Title: Novel natural withanolides induce apoptosis and inhibit migration of neuroblastoma cells through down regulation of N-myc and suppression of Akt/mTOR/NF-κB activation

    doi: 10.18632/oncotarget.24429

    Figure Lengend Snippet: Withanolides induce cellular stress response in NB cells IMR 32 cells were preloaded with ROS indicator CM-H 2 DCFDA prior to withanolides exposure and alteration in oxidation potential was measured. ( A and B ) Fluorescent intensity measurement revelaed the induction of ROS after 4 h of treatment in a dose dependent manner and complete blocking of this effect by 5 mM NAC co-treatment. Immunoblot analysis of the stress response, heat shock proteins demonstrated increased expression levels of HSP32 and HSP70, and decreased expression of HSF1 after 24 h of treatment with increasing concentrations of withanolides. When the cells were pre-treated with NAC, cleavage of PARP that is an indicator of apoptosis was blocked and the increase in the expression levels of the heat shock proteins HSP32 and HSP70 as well as decrease in expression levels of mTOR pathway proteins and Iκ-Bα are blocked after NAC treatment, indicating attenuation of the anti-proliferative properties of withanolides by NAC ( C – E ).

    Article Snippet: Reagents used in the flow cytometry analysis for cell cycle and apoptosis such as propidium idodide (PI), RNase were obtained from Sigma-Aldrich ((St. Louis, MO) and Annexin V-FITC was obtained from BD biosciences (San Diego, CA).

    Techniques: Blocking Assay, Expressing

    Induction of apoptosis after novel withanolides treatment in NB cells ( A and D ) IMR 32 and GOTO cells were stained with PI and annexin V-FITC and then analyzed by flow cytometry after treatment with increasing amounts of each withanolides. ( B and E ) Graphic representation of induction of apoptosis and necrosis after treatment of IMR 32 and GOTO cells with increasing concentrations of withanolides. Mean ± standard deviation results from three independent experiments are shown. ( C and F ) Western blot analysis of cleavage of PARP and caspases 3 and 7 after treatment of NB cells, IMR 32 and GOTO with novel withanolides for 24 h confirms induction of apoptosis. As a loading control actin was used.

    Journal: Oncotarget

    Article Title: Novel natural withanolides induce apoptosis and inhibit migration of neuroblastoma cells through down regulation of N-myc and suppression of Akt/mTOR/NF-κB activation

    doi: 10.18632/oncotarget.24429

    Figure Lengend Snippet: Induction of apoptosis after novel withanolides treatment in NB cells ( A and D ) IMR 32 and GOTO cells were stained with PI and annexin V-FITC and then analyzed by flow cytometry after treatment with increasing amounts of each withanolides. ( B and E ) Graphic representation of induction of apoptosis and necrosis after treatment of IMR 32 and GOTO cells with increasing concentrations of withanolides. Mean ± standard deviation results from three independent experiments are shown. ( C and F ) Western blot analysis of cleavage of PARP and caspases 3 and 7 after treatment of NB cells, IMR 32 and GOTO with novel withanolides for 24 h confirms induction of apoptosis. As a loading control actin was used.

    Article Snippet: Reagents used in the flow cytometry analysis for cell cycle and apoptosis such as propidium idodide (PI), RNase were obtained from Sigma-Aldrich ((St. Louis, MO) and Annexin V-FITC was obtained from BD biosciences (San Diego, CA).

    Techniques: Staining, Flow Cytometry, Cytometry, Standard Deviation, Western Blot

    Concomitant loss of MLKL and Caspase-8, but not loss of RIPK1 kinase activity or combined loss of RIPK3 and Caspase-8, promotes survival of LUBAC-deficient mice a, Representative images of E10.5 ( n =6 embryos/genotype), scale bar: 2 mm, E14.5 ( n =12 Ripk1 K45A Hoil-1 +/- , n =5 Ripk1 K45A Hoil-1 -/- embryos/genotype) and E15.5 embryos ( n =3 embryos/genotype). Scale bar: 5 mm. *: poor yolk sac vascularisation. b, f, Representative images of yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved (cl.) Caspase-3, green) at E14.5 (b) or E13.5 (f) and quantification. Mean ± s.e.m. and P values from unpaired two-tailed t -tests (b) or one-way ANOVA (f) are shown. Scale bar: 50 µm. c, FADD-IP in MEFs treated for 3 h with zVAD-fmk ± TNF ( n . d, Cell death by PI incorporation in MEFs ± TNF (10 ng/ml) or LT-α. Mean ± s.e.m. ( n =3 independent experiments) and P values (**** P

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: Concomitant loss of MLKL and Caspase-8, but not loss of RIPK1 kinase activity or combined loss of RIPK3 and Caspase-8, promotes survival of LUBAC-deficient mice a, Representative images of E10.5 ( n =6 embryos/genotype), scale bar: 2 mm, E14.5 ( n =12 Ripk1 K45A Hoil-1 +/- , n =5 Ripk1 K45A Hoil-1 -/- embryos/genotype) and E15.5 embryos ( n =3 embryos/genotype). Scale bar: 5 mm. *: poor yolk sac vascularisation. b, f, Representative images of yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved (cl.) Caspase-3, green) at E14.5 (b) or E13.5 (f) and quantification. Mean ± s.e.m. and P values from unpaired two-tailed t -tests (b) or one-way ANOVA (f) are shown. Scale bar: 50 µm. c, FADD-IP in MEFs treated for 3 h with zVAD-fmk ± TNF ( n . d, Cell death by PI incorporation in MEFs ± TNF (10 ng/ml) or LT-α. Mean ± s.e.m. ( n =3 independent experiments) and P values (**** P

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Activity Assay, Mouse Assay, Two Tailed Test

    TNFR1 signalling drives cell death and lethality of HOIL-1-deficient mice at mid-gestation. a, d , Quantification of genotypes of animals obtained from inter-crosses of Tnf -/- Hoil-1 +/- (a) and Tnfr1 -/- Hoil-1 +/- (d) mice. *: dead embryos. b, Representative images of embryos quantified in (a) at E10.5 and E15.5, *; poor yolk sac. c, Cell death as detected by whole-mount TUNEL staining in yolk sacs at E10.5 (n= 3 embryos/genotype). e, Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved Caspase-3, bottom panel) of yolk sacs. Merged image is shown in Scale bar: 50 µm. f, Representative images of embryos at E16.5 ( n =2 for Tnfr1 -/- Hoil-1 +/- and n =4 for Tnfr1 -/- Hoil-1 -/- ). Fig. 1g.

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: TNFR1 signalling drives cell death and lethality of HOIL-1-deficient mice at mid-gestation. a, d , Quantification of genotypes of animals obtained from inter-crosses of Tnf -/- Hoil-1 +/- (a) and Tnfr1 -/- Hoil-1 +/- (d) mice. *: dead embryos. b, Representative images of embryos quantified in (a) at E10.5 and E15.5, *; poor yolk sac. c, Cell death as detected by whole-mount TUNEL staining in yolk sacs at E10.5 (n= 3 embryos/genotype). e, Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved Caspase-3, bottom panel) of yolk sacs. Merged image is shown in Scale bar: 50 µm. f, Representative images of embryos at E16.5 ( n =2 for Tnfr1 -/- Hoil-1 +/- and n =4 for Tnfr1 -/- Hoil-1 -/- ). Fig. 1g.

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Mouse Assay, TUNEL Assay, Staining

    HOIL-1-deficient mice die at mid-gestation a, Schematic representation of the Hoil-1 knockout strategy. Solid boxes represent Hoil-1 exons and grey boxes with a star indicate the targeted exons. Boxes with diagonal and horizontal strips represent LoxP and Frt sites, respectively. b, Specificity of gene recombination was assessed by Southern blotting with 5’ and 3’ probes external to the construct in four clones (14B8, 14F6, 20D7 and 21F7). Digest of the DNA with ApaI, followed by hybridisation with the 3’ probe was expected to show a 5700 bp band for the WT allele and a 7700 bp band for the mutant allele. All four clones appeared to have the correct recombination on the 3’ side. Digest of the DNA with SphI and hybridisation with the 5’ probe was expected to show a 4500 bp WT band and a 6200 bp band for the mutated allele. Clones 14B8, 14F6 and 21F7 appeared to be correctly recombined on the 5’ side. Finally, cutting the DNA with ApaI and hybridising with a hygro probe showed a single band in all clones, indicative of a single integration of the construct in all four ES clones. Clones 14B8 and 14F6 were selected for generation of the two Hoil-1 -/- strains. c , PCR analysis of Hoil-1 wild-type, heterozygous and knockout mice. d , Protein levels of HOIL-1, HOIP and SHARPIN in whole embryo lysates ( n = 3 for Hoil-1 +/- and Hoil-1 -/- embryos and n =1 for Hoil-1 +/+ . e , Quantification of genotypes of animals obtained from inter-crossing C20Hoil-1 +/- mice. *indicates dead embryos. f , Representative images of C20Hoil-1 +/- and C20Hoil-1 -/- embryos from E9.5 to E11.5 as quantified in (e). Scale bar: 2 mm. g, Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved Caspase-3, bottom panel) of yolk sacs. Merged image is shown in . h , Whole-mount TUNEL staining of embryos at the indicated stages (embryo/genotype n =2 at E10.5, n =8 for Hoil-1 +/- and n =5 for Hoil-1 -/- at E11.5). Scale bar: 2 mm. i, Quantification of genotypes of animals obtained from inter-crossing Hoil-1 fl/wt Tie2-Cre + with Hoil-1 fl/fl Tie2-Cre - mice. *indicates dead embryos. j , Representative images of embryos with conditional deletion of Hoil-1 in Tie2-Cre expressing cells as quantified in (i). Scale bar: 2 mm. *: poorly vascularised yolk sac. Fig. 1c

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: HOIL-1-deficient mice die at mid-gestation a, Schematic representation of the Hoil-1 knockout strategy. Solid boxes represent Hoil-1 exons and grey boxes with a star indicate the targeted exons. Boxes with diagonal and horizontal strips represent LoxP and Frt sites, respectively. b, Specificity of gene recombination was assessed by Southern blotting with 5’ and 3’ probes external to the construct in four clones (14B8, 14F6, 20D7 and 21F7). Digest of the DNA with ApaI, followed by hybridisation with the 3’ probe was expected to show a 5700 bp band for the WT allele and a 7700 bp band for the mutant allele. All four clones appeared to have the correct recombination on the 3’ side. Digest of the DNA with SphI and hybridisation with the 5’ probe was expected to show a 4500 bp WT band and a 6200 bp band for the mutated allele. Clones 14B8, 14F6 and 21F7 appeared to be correctly recombined on the 5’ side. Finally, cutting the DNA with ApaI and hybridising with a hygro probe showed a single band in all clones, indicative of a single integration of the construct in all four ES clones. Clones 14B8 and 14F6 were selected for generation of the two Hoil-1 -/- strains. c , PCR analysis of Hoil-1 wild-type, heterozygous and knockout mice. d , Protein levels of HOIL-1, HOIP and SHARPIN in whole embryo lysates ( n = 3 for Hoil-1 +/- and Hoil-1 -/- embryos and n =1 for Hoil-1 +/+ . e , Quantification of genotypes of animals obtained from inter-crossing C20Hoil-1 +/- mice. *indicates dead embryos. f , Representative images of C20Hoil-1 +/- and C20Hoil-1 -/- embryos from E9.5 to E11.5 as quantified in (e). Scale bar: 2 mm. g, Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved Caspase-3, bottom panel) of yolk sacs. Merged image is shown in . h , Whole-mount TUNEL staining of embryos at the indicated stages (embryo/genotype n =2 at E10.5, n =8 for Hoil-1 +/- and n =5 for Hoil-1 -/- at E11.5). Scale bar: 2 mm. i, Quantification of genotypes of animals obtained from inter-crossing Hoil-1 fl/wt Tie2-Cre + with Hoil-1 fl/fl Tie2-Cre - mice. *indicates dead embryos. j , Representative images of embryos with conditional deletion of Hoil-1 in Tie2-Cre expressing cells as quantified in (i). Scale bar: 2 mm. *: poorly vascularised yolk sac. Fig. 1c

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Mouse Assay, Knock-Out, Southern Blot, Construct, Clone Assay, Hybridization, Mutagenesis, Polymerase Chain Reaction, Staining, TUNEL Assay, Expressing

    Individual deletion of mediators of apoptosis or necroptosis does not prevent cell death and lethality at mid-gestation of HOIL-1- or HOIP-deficient embryos. a , Western blot analysis of MLKL expression in the indicated organs derived from control Mlkl -/- mice ( n . b, d, e, f, Representative images of embryos at different stages of gestation (E10.5: n =7 for Ripk3 -/- Hoil-1 +/- and n =5 for Ripk3 -/- Hoil-1 -/- ; E11.5: n =5 for Ripk3 -/- Hoil-1 +/- and n =2 for Ripk3 -/- Hoil-1 -/- ; E12.5: n =9 for Ripk3 -/- Hoil-1 +/- and n =2 for Ripk3 -/- Hoil-1 -/- (b), E10.5: n =16 for Mlkl -/- Hoip +/- and n =6 for Mlkl -/- Hoip -/- ; E11.5: n =8 for Mlkl -/- Hoip +/- and n =6 for Mlkl -/- Hoip -/- ; E12.5: n =10 for Mlkl -/- Hoip +/- and n =5 for Mlkl -/- Hoip -/- (d), E10.5: n =5 for Caspase-8 +/- Hoip +/- and n =4 for Caspase-8 +/- Hoip -/- ; E11.5: n =6 for Caspase-8 +/- Hoip +/- and n =3 for Caspase-8 +/- Hoip -/- ; E12.5: n =3 for Caspase-8 +/- Hoip +/- and n =2 for Caspase-8 +/- Hoip -/- (e), E10.5: n =2 for Caspase-8 +/- Hoil-1 +/- and n =4 for Caspase-8 +/- Hoil-1 -/- ; E11.5: n =2 for Caspase-8 +/- Hoil-1 +/- and n =5 for Caspase-8 +/- Hoil-1 -/- ; E12.5: n =6 for Caspase-8 +/- Hoil-1 +/- and n =3 for Caspase-8 +/- Hoil-1 -/- (f)). *: poor yolk sac vascularisation. Scale bar: 2 mm. c, Representative images of yolk sac vascularisation and cell death at E10.5 as detected by PECAM-1 (red) and cleaved (cl.) Caspase-3 staining (green) (top panel) and whole mount TUNEL staining (bottom panel) ( n =4 per genotype). Scale bar: 50 µm.

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: Individual deletion of mediators of apoptosis or necroptosis does not prevent cell death and lethality at mid-gestation of HOIL-1- or HOIP-deficient embryos. a , Western blot analysis of MLKL expression in the indicated organs derived from control Mlkl -/- mice ( n . b, d, e, f, Representative images of embryos at different stages of gestation (E10.5: n =7 for Ripk3 -/- Hoil-1 +/- and n =5 for Ripk3 -/- Hoil-1 -/- ; E11.5: n =5 for Ripk3 -/- Hoil-1 +/- and n =2 for Ripk3 -/- Hoil-1 -/- ; E12.5: n =9 for Ripk3 -/- Hoil-1 +/- and n =2 for Ripk3 -/- Hoil-1 -/- (b), E10.5: n =16 for Mlkl -/- Hoip +/- and n =6 for Mlkl -/- Hoip -/- ; E11.5: n =8 for Mlkl -/- Hoip +/- and n =6 for Mlkl -/- Hoip -/- ; E12.5: n =10 for Mlkl -/- Hoip +/- and n =5 for Mlkl -/- Hoip -/- (d), E10.5: n =5 for Caspase-8 +/- Hoip +/- and n =4 for Caspase-8 +/- Hoip -/- ; E11.5: n =6 for Caspase-8 +/- Hoip +/- and n =3 for Caspase-8 +/- Hoip -/- ; E12.5: n =3 for Caspase-8 +/- Hoip +/- and n =2 for Caspase-8 +/- Hoip -/- (e), E10.5: n =2 for Caspase-8 +/- Hoil-1 +/- and n =4 for Caspase-8 +/- Hoil-1 -/- ; E11.5: n =2 for Caspase-8 +/- Hoil-1 +/- and n =5 for Caspase-8 +/- Hoil-1 -/- ; E12.5: n =6 for Caspase-8 +/- Hoil-1 +/- and n =3 for Caspase-8 +/- Hoil-1 -/- (f)). *: poor yolk sac vascularisation. Scale bar: 2 mm. c, Representative images of yolk sac vascularisation and cell death at E10.5 as detected by PECAM-1 (red) and cleaved (cl.) Caspase-3 staining (green) (top panel) and whole mount TUNEL staining (bottom panel) ( n =4 per genotype). Scale bar: 50 µm.

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Western Blot, Expressing, Derivative Assay, Mouse Assay, Staining, TUNEL Assay

    Combined deletion of MLKL and Caspase-8 promotes survival of LUBAC-deficient mice. a, Quantification of genotypes of animals obtained from inter-crosses of Mlkl -/- Caspase-8 +/- Hoip +/- with Mlkl -/- Caspase-8 -/- Hoip +/- mice. *: dead embryos. b, Representative images of adult mice as quantified in (a). c , Kaplan-Meier plot of mouse survival ( n =6 for Mlkl -/- Caspase-8 -/- Hoip -/- and n =9 for Mlkl -/- Caspase-8 -/- Hoil-1 -/- mice). d, Representative images of H E staining of the indicated organs ( n =3 mice/genotype). Scale bar: 200 µm. e, Representative images of yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved (cl.) Caspase-3, green) (top panel) at E13.5 and respective quantifications (bottom panel). Mean ± s.e.m ( n =5 for Mlkl -/- Caspase-8 -/- Hoil-1 +/- and Mlkl -/- Caspase-8 -/- Hoil-1 -/- and n =2 for Mlkl -/- Caspase-8 +/- Hoil-1 -/- ) are shown and results were analysed with unpaired two-tailed t -tests comparing Mlkl -/- Caspase-8 -/- Hoil-1 +/- and Mlkl -/- Caspase-8 -/- Hoil-1 -/- embryos. f, Representative images of H E staining of the indicated organs ( n =3 embryos/genotype). Scale bar: 200 µm. g, Epidermal thickness quantification of mice of the indicated genotypes in (f). Mean ± s.e.m values ( n =3 mice/genotype) are shown and results were analysed with unpaired two-tailed t -tests. h, Western blot analysis of lysates from whole E13.5 embryos of the indicated genotypes as well as L929 cells treated or not with TNF plus zVAD-fmk for 2 h as antibody validation ( n .

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: Combined deletion of MLKL and Caspase-8 promotes survival of LUBAC-deficient mice. a, Quantification of genotypes of animals obtained from inter-crosses of Mlkl -/- Caspase-8 +/- Hoip +/- with Mlkl -/- Caspase-8 -/- Hoip +/- mice. *: dead embryos. b, Representative images of adult mice as quantified in (a). c , Kaplan-Meier plot of mouse survival ( n =6 for Mlkl -/- Caspase-8 -/- Hoip -/- and n =9 for Mlkl -/- Caspase-8 -/- Hoil-1 -/- mice). d, Representative images of H E staining of the indicated organs ( n =3 mice/genotype). Scale bar: 200 µm. e, Representative images of yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved (cl.) Caspase-3, green) (top panel) at E13.5 and respective quantifications (bottom panel). Mean ± s.e.m ( n =5 for Mlkl -/- Caspase-8 -/- Hoil-1 +/- and Mlkl -/- Caspase-8 -/- Hoil-1 -/- and n =2 for Mlkl -/- Caspase-8 +/- Hoil-1 -/- ) are shown and results were analysed with unpaired two-tailed t -tests comparing Mlkl -/- Caspase-8 -/- Hoil-1 +/- and Mlkl -/- Caspase-8 -/- Hoil-1 -/- embryos. f, Representative images of H E staining of the indicated organs ( n =3 embryos/genotype). Scale bar: 200 µm. g, Epidermal thickness quantification of mice of the indicated genotypes in (f). Mean ± s.e.m values ( n =3 mice/genotype) are shown and results were analysed with unpaired two-tailed t -tests. h, Western blot analysis of lysates from whole E13.5 embryos of the indicated genotypes as well as L929 cells treated or not with TNF plus zVAD-fmk for 2 h as antibody validation ( n .

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Mouse Assay, Staining, Two Tailed Test, Western Blot

    Combined deletion of RIPK3 and Caspase-8 prevents cell death but not embryonic lethality at late gestation that is caused by the loss of HOIL-1. a , Quantification of genotypes of animals obtained from inter-crosses of Ripk3 -/- Caspase-8 +/- Hoil-1 +/- with Ripk3 -/- Caspase-8 -/- Hoil-1 +/- mice (left panel) or Ripk3 -/- Caspase-8 -/- Hoil-1 +/- mice (right panel). b, Health status of Ripk3 -/- Caspase-8 +/- Hoil-1 -/- and Ripk3 -/- Caspase-8 -/- Hoil-1 -/- embryos at different developmental stages. c , Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved (cl.) Caspase-3, bottom panel) of yolk sacs. Merged image is shown in . Scale bar: 50 µm. d, Cell death as detected by whole-mount TUNEL staining in yolk sacs at E14.5 (left panel) and respective quantification (right panel). Mean ± s.e.m. ( n =3 embryos/genotype) and P values from one-way ANOVA are reported. e, f Representative images (e) and quantification (f) of cell death in different organs as detected by TUNEL staining at E13.5 ( n =3 embryos/genotype) and E14.5 ( n =5 for Ripk3 -/- Caspase-8 -/- Hoil-1 +/- , n=2 for Ripk3 -/- Caspase-8 -/- Hoil-1 -/- and Ripk3 -/- Caspase-8 -/- Hoil-1 -/- lung and liver and n =3 Ripk3 -/- Caspase-8 -/- Hoil-1 -/- heart). Scale bar: 50 µm (e). Mean ± s.e.m. values are shown (f). g, Cell death was analysed by PI staining in MEFs stimulated or not with the indicated ligands for 24 h. Mean ± s.e.m. ( n =3 independent experiments) and P values from two-way ANOVA are reported. h, Representative images of H E staining on E13.5 whole-embryo paraffin embedded sections ( n =3 for Ripk3 -/- Caspase-8 -/- Hoil-1 +/- and Ripk3 -/- Caspase-8 -/- Hoil-1 -/- and n =2 for Ripk3 -/- Caspase-8 +/- Hoil-1 -/- ). *: pericardial effusion, arrows; congested vessels H, heart; L, lung; Li, liver. Scale bar: 200 µm. i, Representative images of micro-focus CT scan images of whole E13.5 embryos ( n =3 embryos/genotype). *: pericardial effusion Fig. 3f

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: Combined deletion of RIPK3 and Caspase-8 prevents cell death but not embryonic lethality at late gestation that is caused by the loss of HOIL-1. a , Quantification of genotypes of animals obtained from inter-crosses of Ripk3 -/- Caspase-8 +/- Hoil-1 +/- with Ripk3 -/- Caspase-8 -/- Hoil-1 +/- mice (left panel) or Ripk3 -/- Caspase-8 -/- Hoil-1 +/- mice (right panel). b, Health status of Ripk3 -/- Caspase-8 +/- Hoil-1 -/- and Ripk3 -/- Caspase-8 -/- Hoil-1 -/- embryos at different developmental stages. c , Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved (cl.) Caspase-3, bottom panel) of yolk sacs. Merged image is shown in . Scale bar: 50 µm. d, Cell death as detected by whole-mount TUNEL staining in yolk sacs at E14.5 (left panel) and respective quantification (right panel). Mean ± s.e.m. ( n =3 embryos/genotype) and P values from one-way ANOVA are reported. e, f Representative images (e) and quantification (f) of cell death in different organs as detected by TUNEL staining at E13.5 ( n =3 embryos/genotype) and E14.5 ( n =5 for Ripk3 -/- Caspase-8 -/- Hoil-1 +/- , n=2 for Ripk3 -/- Caspase-8 -/- Hoil-1 -/- and Ripk3 -/- Caspase-8 -/- Hoil-1 -/- lung and liver and n =3 Ripk3 -/- Caspase-8 -/- Hoil-1 -/- heart). Scale bar: 50 µm (e). Mean ± s.e.m. values are shown (f). g, Cell death was analysed by PI staining in MEFs stimulated or not with the indicated ligands for 24 h. Mean ± s.e.m. ( n =3 independent experiments) and P values from two-way ANOVA are reported. h, Representative images of H E staining on E13.5 whole-embryo paraffin embedded sections ( n =3 for Ripk3 -/- Caspase-8 -/- Hoil-1 +/- and Ripk3 -/- Caspase-8 -/- Hoil-1 -/- and n =2 for Ripk3 -/- Caspase-8 +/- Hoil-1 -/- ). *: pericardial effusion, arrows; congested vessels H, heart; L, lung; Li, liver. Scale bar: 200 µm. i, Representative images of micro-focus CT scan images of whole E13.5 embryos ( n =3 embryos/genotype). *: pericardial effusion Fig. 3f

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Mouse Assay, Staining, TUNEL Assay, Computed Tomography

    Ablation of the kinase activity of RIPK1 in HOIL-1- or HOIP-deficient embryos prevents cell death and lethality at mid-gestation but not at late gestation. a, b, Quantification of genotypes of animals obtained after inter-crossing Ripk1 K45A Hoil-1 +/- (a) and Ripk1 K45A Hoip +/- (b) mice. *indicates dead embryos. c, Representative images of embryos quantified in (b) *; poor yolk sac vascularisation. Scale bar: 2 mm. d, Whole-mount TUNEL staining of embryos ( n =2 embryos). Scale bar: 2 mm. e, Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved (cl.) Caspase-3, bottom panel) of yolk sacs. Merged image is shown in . f, g, Representative images of cell death in different organs (f) and quantification (g) as detected by TUNEL staining at E14.5 ( n =3 embryos/genotype). Scale bar: 50 μm (f). Mean ± s.e.m. ( n =3 embryos/genotype) and P values from one-way ANOVA are reported (g). h, Representative images of H E staining on whole-embryo paraffin sections ( n =3 embryos/genotype). *; pericardial effusion, n, necrotic area. H, heart; L, lung; Li, liver. Scale bar: 200 µm. i, Cell death was analysed by PI staining in MEFs stimulated or not with TNF for 24 h plus the indicated cell death inhibitors. Mean ± s.e.m. ( n =3 independent experiments) and P values from two-way ANOVA are reported. Fig. 3b

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: Ablation of the kinase activity of RIPK1 in HOIL-1- or HOIP-deficient embryos prevents cell death and lethality at mid-gestation but not at late gestation. a, b, Quantification of genotypes of animals obtained after inter-crossing Ripk1 K45A Hoil-1 +/- (a) and Ripk1 K45A Hoip +/- (b) mice. *indicates dead embryos. c, Representative images of embryos quantified in (b) *; poor yolk sac vascularisation. Scale bar: 2 mm. d, Whole-mount TUNEL staining of embryos ( n =2 embryos). Scale bar: 2 mm. e, Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved (cl.) Caspase-3, bottom panel) of yolk sacs. Merged image is shown in . f, g, Representative images of cell death in different organs (f) and quantification (g) as detected by TUNEL staining at E14.5 ( n =3 embryos/genotype). Scale bar: 50 μm (f). Mean ± s.e.m. ( n =3 embryos/genotype) and P values from one-way ANOVA are reported (g). h, Representative images of H E staining on whole-embryo paraffin sections ( n =3 embryos/genotype). *; pericardial effusion, n, necrotic area. H, heart; L, lung; Li, liver. Scale bar: 200 µm. i, Cell death was analysed by PI staining in MEFs stimulated or not with TNF for 24 h plus the indicated cell death inhibitors. Mean ± s.e.m. ( n =3 independent experiments) and P values from two-way ANOVA are reported. Fig. 3b

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Activity Assay, Mouse Assay, TUNEL Assay, Staining

    HOIL-1 deficiency causes embryonic lethality at mid-gestation due to TNFR1-mediated endothelial cell death a, Mendelian frequencies obtained from inter-crossing Hoil-1 +/- mice, *: dead embryos. b, Representative images of embryos from E9.5 to E11.5 quantified in (a), *: poor yolk sac vascularisation. Scale bar: 2 mm. c, Representative images of yolk sac vascularisation (PECAM-1, red) and cell death (cleaved (cl.) Caspase-3 staining, green) at E10.5 (top panel) ( n =4 yolk-sacs/genotype), and whole-mount TUNEL staining (bottom, panel) ( n =2 yolk-sacs/genotype). Scale bar: 50 µm. d, g, Quantification of branching points (g) and cleaved Caspase-3 positive cells (g). Mean ± s.e.m. values and P values from unpaired two-tailed t -tests are shown. e, Representative images of embryos at E10.5 ( n =14 Hoil-1 fl/wt Tie2-Cre+ and n =7 Hoil-1 fl/fl Tie2-Cre+ embryos, top panel). *: poor yolk sac vascularisation. Scale bar: 2 mm. Yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved Caspase-3, green) (middle panel). Scale bar: 50 µm. Yolk sac whole-mount TUNEL staining ( n =6 Hoil-1 fl/wt Tie2-Cre+ and n =2 Hoil-1 fl/fl Tie2-Cre+ yolk-sacs/genotype , bottom panel). f , Representative images of embryos at E15.5 (top panel, n =6 Tnfr1 -/- Hoil-1 -/- and n =19 Tnfr1 -/- Hoil-1 +/- embryos), scale bar: 2 mm, and yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved Caspase-3, green) (bottom panel), Scale bar: 50 µm. h, Representative images of H E staining on whole-embryo paraffin sections ( n =3 embryos/genotype). *: pericardial effusion, arrows; congested vessels. H, heart; L, lung; Li, liver. Scale bar: 50 µm.

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: HOIL-1 deficiency causes embryonic lethality at mid-gestation due to TNFR1-mediated endothelial cell death a, Mendelian frequencies obtained from inter-crossing Hoil-1 +/- mice, *: dead embryos. b, Representative images of embryos from E9.5 to E11.5 quantified in (a), *: poor yolk sac vascularisation. Scale bar: 2 mm. c, Representative images of yolk sac vascularisation (PECAM-1, red) and cell death (cleaved (cl.) Caspase-3 staining, green) at E10.5 (top panel) ( n =4 yolk-sacs/genotype), and whole-mount TUNEL staining (bottom, panel) ( n =2 yolk-sacs/genotype). Scale bar: 50 µm. d, g, Quantification of branching points (g) and cleaved Caspase-3 positive cells (g). Mean ± s.e.m. values and P values from unpaired two-tailed t -tests are shown. e, Representative images of embryos at E10.5 ( n =14 Hoil-1 fl/wt Tie2-Cre+ and n =7 Hoil-1 fl/fl Tie2-Cre+ embryos, top panel). *: poor yolk sac vascularisation. Scale bar: 2 mm. Yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved Caspase-3, green) (middle panel). Scale bar: 50 µm. Yolk sac whole-mount TUNEL staining ( n =6 Hoil-1 fl/wt Tie2-Cre+ and n =2 Hoil-1 fl/fl Tie2-Cre+ yolk-sacs/genotype , bottom panel). f , Representative images of embryos at E15.5 (top panel, n =6 Tnfr1 -/- Hoil-1 -/- and n =19 Tnfr1 -/- Hoil-1 +/- embryos), scale bar: 2 mm, and yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved Caspase-3, green) (bottom panel), Scale bar: 50 µm. h, Representative images of H E staining on whole-embryo paraffin sections ( n =3 embryos/genotype). *: pericardial effusion, arrows; congested vessels. H, heart; L, lung; Li, liver. Scale bar: 50 µm.

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Mouse Assay, Staining, TUNEL Assay, Two Tailed Test