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    Enzo Biochem parp 1
    Chip-seq analysis of HIF-1α capacity to bind the promoters of its target genes on WT vs. <t>PARP-1</t> KO cells. a ChIP-Seq performed for HIF-1α after 4 h of hypoxia on HEK 293T and PARP-1 K.O cells. The HIF-1α binding sites are presented considering the function of the closest gene. b Representation of the percentage of loss on the different functions on the PARP-1 KO cells. c MEME analysis showing the most common sequences where HIF-1α binds and their E-value.
    Parp 1, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/parp 1/product/Enzo Biochem
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    parp 1 - by Bioz Stars, 2022-09
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    99
    Becton Dickinson anti ripk1
    Cytotoxic TNF and TRAIL signaling in <t>FADD-RIPK1</t> and FADD-TRADD double-deficient HeLa-RIPK3 cells. a Western blot evaluation of TRADD, RIPK1, and FADD expression of HeLa-RIPK3 con , HeLa-RIPK3-FADD/RIPK1 DKO and HeLa-RIPK3-FADD/TRADD DKO cells. b Cells were sensitized with 2.5 µg/ml CHX and were stimulated overnight in technical triplicates with the indicated combinations of TNF, TRAIL, ZVAD (20 µM), and nec1 (90 µM). Cellular viability was finally determined by crystal violet staining. A representative panel of experiments is shown. For statistical analysis of independent experiments please see Tables 1 and 2 and Supplementary Tables I – III . c Cells were sensitized with 2.5 µg/ml CHX and stimulated with 100 ng/ml TNF or 100 ng/ml TRAIL for 0–6 h. Total cell lysates were analyzed by western blot for processing of the indicated caspases and caspase substrates. fl full-length
    Anti Ripk1, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti ripk1/product/Becton Dickinson
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti ripk1 - by Bioz Stars, 2022-09
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    Image Search Results


    Chip-seq analysis of HIF-1α capacity to bind the promoters of its target genes on WT vs. PARP-1 KO cells. a ChIP-Seq performed for HIF-1α after 4 h of hypoxia on HEK 293T and PARP-1 K.O cells. The HIF-1α binding sites are presented considering the function of the closest gene. b Representation of the percentage of loss on the different functions on the PARP-1 KO cells. c MEME analysis showing the most common sequences where HIF-1α binds and their E-value.

    Journal: Redox Biology

    Article Title: Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

    doi: 10.1016/j.redox.2021.101885

    Figure Lengend Snippet: Chip-seq analysis of HIF-1α capacity to bind the promoters of its target genes on WT vs. PARP-1 KO cells. a ChIP-Seq performed for HIF-1α after 4 h of hypoxia on HEK 293T and PARP-1 K.O cells. The HIF-1α binding sites are presented considering the function of the closest gene. b Representation of the percentage of loss on the different functions on the PARP-1 KO cells. c MEME analysis showing the most common sequences where HIF-1α binds and their E-value.

    Article Snippet: In the present work we connect both pathways through HIF-1α C-terminal PARylation via PARP-1.

    Techniques: Chromatin Immunoprecipitation, Binding Assay

    During hypoxia, ROS production leads to PARP-1 activation. This protein interacts with HIF-1α at its C-terminus domain, causing its PTM with poly(ADP-ribose). This modification leads to HIF-1α accumulation. HIF-1α then binds to the promoters of its target genes, causing the expression of hypoxic genes and allowing the cell to adapt to hypoxia. On the other hand, when PARP-1 is inhibited or knocked out, it causes HIF-1α downregulation. This reduces HIF-1α binding to its promoters causing a reduced hypoxic gene expression. These changes lead to a poorer adaptation to hypoxia.

    Journal: Redox Biology

    Article Title: Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

    doi: 10.1016/j.redox.2021.101885

    Figure Lengend Snippet: During hypoxia, ROS production leads to PARP-1 activation. This protein interacts with HIF-1α at its C-terminus domain, causing its PTM with poly(ADP-ribose). This modification leads to HIF-1α accumulation. HIF-1α then binds to the promoters of its target genes, causing the expression of hypoxic genes and allowing the cell to adapt to hypoxia. On the other hand, when PARP-1 is inhibited or knocked out, it causes HIF-1α downregulation. This reduces HIF-1α binding to its promoters causing a reduced hypoxic gene expression. These changes lead to a poorer adaptation to hypoxia.

    Article Snippet: In the present work we connect both pathways through HIF-1α C-terminal PARylation via PARP-1.

    Techniques: Activation Assay, Modification, Expressing, Binding Assay

    a . On 293T WT and PARP-1 KO, pie charts presenting the porcentaje of HIF-1α binding sites detected on different areas of the gene structure. b Percentaje of HIF-1α accumulation on the genome regarding the distance (measured as kilobases) from the TSS. c Study of the promoter area (showing 2 kb around the TSS), presenting the frequency of HIF-1α detection on 293T WT and PARP-1 KO cells during hypoxia (1% O 2 , 4 h).

    Journal: Redox Biology

    Article Title: Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

    doi: 10.1016/j.redox.2021.101885

    Figure Lengend Snippet: a . On 293T WT and PARP-1 KO, pie charts presenting the porcentaje of HIF-1α binding sites detected on different areas of the gene structure. b Percentaje of HIF-1α accumulation on the genome regarding the distance (measured as kilobases) from the TSS. c Study of the promoter area (showing 2 kb around the TSS), presenting the frequency of HIF-1α detection on 293T WT and PARP-1 KO cells during hypoxia (1% O 2 , 4 h).

    Article Snippet: In the present work we connect both pathways through HIF-1α C-terminal PARylation via PARP-1.

    Techniques: Binding Assay

    Impact of PARP-1 inhibition/knockout on cellular metabolism and fitness. a Oxygen consumption analysis. HEK 293T WT cells and HEK 293T PARP-1 KO were exposed to normoxia or 4 h of hypoxia. Then fluorescent emission was measured and compared during 60 min via fluorescence spectroscopy. b Glycolysis assay measured as cytoplasmic acidification. HEK 293T WT cells and HEK 293T PARP-1 KO were exposed to 4 h of normoxia and hypoxia, following glycolysis measurement for 60 min via fluorescent emission. Antimycin A was used as a positive control. c Cell growth during different times of normoxia and hypoxia. HEK 293T cells undergoing PARP inhibition (olaparib 5 μM) or PARP-1 KO cells. 24 h after culture, cells were treated and exposed to 1% hypoxia during crescent times. d Wound healing assay performed on HEK 293T cells, being PARP inhibited (olaparib 5 μM) or PARP-1 KO, wound healing measurement was performed 24 h after wound opening.

    Journal: Redox Biology

    Article Title: Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

    doi: 10.1016/j.redox.2021.101885

    Figure Lengend Snippet: Impact of PARP-1 inhibition/knockout on cellular metabolism and fitness. a Oxygen consumption analysis. HEK 293T WT cells and HEK 293T PARP-1 KO were exposed to normoxia or 4 h of hypoxia. Then fluorescent emission was measured and compared during 60 min via fluorescence spectroscopy. b Glycolysis assay measured as cytoplasmic acidification. HEK 293T WT cells and HEK 293T PARP-1 KO were exposed to 4 h of normoxia and hypoxia, following glycolysis measurement for 60 min via fluorescent emission. Antimycin A was used as a positive control. c Cell growth during different times of normoxia and hypoxia. HEK 293T cells undergoing PARP inhibition (olaparib 5 μM) or PARP-1 KO cells. 24 h after culture, cells were treated and exposed to 1% hypoxia during crescent times. d Wound healing assay performed on HEK 293T cells, being PARP inhibited (olaparib 5 μM) or PARP-1 KO, wound healing measurement was performed 24 h after wound opening.

    Article Snippet: In the present work we connect both pathways through HIF-1α C-terminal PARylation via PARP-1.

    Techniques: Inhibition, Knock-Out, Fluorescence, Spectroscopy, Positive Control, Wound Healing Assay

    a Correlation of gene In vivo study of PARP1 and HIF-1α expression in melanoma patients using the publicly available database cBioportal; top panel, metastatic melanoma (CM Spearman: 0.50 (p = 8.722e-3)Pearson:0.42 (p = 0.0309)); middle panel, uveal melanoma (UM Spearman: 0.30 (p = 6.295e-3), Pearson: 0.33 (p = 2.655e-3)); lower panel, acral melanoma AM Spearman: 0.55 (p = 4.636e-4)Pearson: 0.59 (p = 1.516e-4)). b Tissue array on 3 melanoma patients, immunochemistry analysis is performed to observe PARP-1 and HIF1α expression. Consecutive sections are shown. c Three different cell lines (MUM2B, C8161 and Hela) show a reduction on HIF-1α after PARP inhibition using Olaparib 5 μM during hypoxia 4 h. d Transient silencing of PARP-1 on HEK 293T cells shows an impairment on HIF-1α accumulation during early (4 h) hypoxia. e In Hela cells polymer accumulation is induced on PARG silenced cell. After 4 h of hypoxia HIF-1α is more stable on the polymer enhanced context.

    Journal: Redox Biology

    Article Title: Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

    doi: 10.1016/j.redox.2021.101885

    Figure Lengend Snippet: a Correlation of gene In vivo study of PARP1 and HIF-1α expression in melanoma patients using the publicly available database cBioportal; top panel, metastatic melanoma (CM Spearman: 0.50 (p = 8.722e-3)Pearson:0.42 (p = 0.0309)); middle panel, uveal melanoma (UM Spearman: 0.30 (p = 6.295e-3), Pearson: 0.33 (p = 2.655e-3)); lower panel, acral melanoma AM Spearman: 0.55 (p = 4.636e-4)Pearson: 0.59 (p = 1.516e-4)). b Tissue array on 3 melanoma patients, immunochemistry analysis is performed to observe PARP-1 and HIF1α expression. Consecutive sections are shown. c Three different cell lines (MUM2B, C8161 and Hela) show a reduction on HIF-1α after PARP inhibition using Olaparib 5 μM during hypoxia 4 h. d Transient silencing of PARP-1 on HEK 293T cells shows an impairment on HIF-1α accumulation during early (4 h) hypoxia. e In Hela cells polymer accumulation is induced on PARG silenced cell. After 4 h of hypoxia HIF-1α is more stable on the polymer enhanced context.

    Article Snippet: In the present work we connect both pathways through HIF-1α C-terminal PARylation via PARP-1.

    Techniques: In Vivo, Expressing, Inhibition

    PARP-1 physically interacts and PARylates HIF-1α at its C-Ter domain. a Immunoprecipitation of HIF-1α on HEK 293T cells undergoing normoxia and hypoxia 4 h. A complex is form leading to PARylation. b Immunoprecipitation of HIF-1α and PARP-1 on HEK 293T cells exposed to normoxia or hypoxia 4 h. During PARP-1 inhibition using PJ34 10 μM the complex is destabilized. c Immunoprecipitation of PAR polymer on HEK 293T cells transfected with the C-ter or DMS domain of HIF-1α during normoxia and early hypoxia 4 h. During PARP inhibition with PJ34 10 μM the complex observed between PARP-1 and the endogenous HIF-1α or the C-ter is lost. d In vitro PARylation assay for PARP-1 and HIF-1α. Coomasie staining and 32 P-NAD-PAR autoradiography is presented. e Synthesis of four peptides located on the HIF-1α C-ter. Two WT and their correspondent non PARylable analogs are presented. f in vitro PARylation assay performed in the four peptides. The results are presented in counts per minute.

    Journal: Redox Biology

    Article Title: Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

    doi: 10.1016/j.redox.2021.101885

    Figure Lengend Snippet: PARP-1 physically interacts and PARylates HIF-1α at its C-Ter domain. a Immunoprecipitation of HIF-1α on HEK 293T cells undergoing normoxia and hypoxia 4 h. A complex is form leading to PARylation. b Immunoprecipitation of HIF-1α and PARP-1 on HEK 293T cells exposed to normoxia or hypoxia 4 h. During PARP-1 inhibition using PJ34 10 μM the complex is destabilized. c Immunoprecipitation of PAR polymer on HEK 293T cells transfected with the C-ter or DMS domain of HIF-1α during normoxia and early hypoxia 4 h. During PARP inhibition with PJ34 10 μM the complex observed between PARP-1 and the endogenous HIF-1α or the C-ter is lost. d In vitro PARylation assay for PARP-1 and HIF-1α. Coomasie staining and 32 P-NAD-PAR autoradiography is presented. e Synthesis of four peptides located on the HIF-1α C-ter. Two WT and their correspondent non PARylable analogs are presented. f in vitro PARylation assay performed in the four peptides. The results are presented in counts per minute.

    Article Snippet: In the present work we connect both pathways through HIF-1α C-terminal PARylation via PARP-1.

    Techniques: Immunoprecipitation, Inhibition, Transfection, In Vitro, Staining, Autoradiography

    HIF-1α interacts with PARP-1 at its C-terminus domain regulating its stability. a HIF-1α constructs presenting the wild type sequences for the HIF-1α wt, short WT and C-ter. The PHD residues mutated are presented in blue, obtaining the DML and DMS. b-f On HEK 293T western blots showing the downregulation during PHD overexpression of the HIF-1α and short wt. This reduction is impaired on the PHD insensitive mutants. g Western blot study on HEK 293T of the three PHD-insensitive domains of HIF-1α, their stability is compared during normoxia and hypoxia 4 h with PARP activated or inhibited using PJ34 10 μM h PARP-1 domains presenting the full protein, and separately, the DNA-binding, the automodification and the catalytic domain. i pull-Down assay exposing the different domains of PARP-1 to the C-ter domain of HIF-1α. The C-ter binds to PARP-1 full protein and the auto modification domain.

    Journal: Redox Biology

    Article Title: Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

    doi: 10.1016/j.redox.2021.101885

    Figure Lengend Snippet: HIF-1α interacts with PARP-1 at its C-terminus domain regulating its stability. a HIF-1α constructs presenting the wild type sequences for the HIF-1α wt, short WT and C-ter. The PHD residues mutated are presented in blue, obtaining the DML and DMS. b-f On HEK 293T western blots showing the downregulation during PHD overexpression of the HIF-1α and short wt. This reduction is impaired on the PHD insensitive mutants. g Western blot study on HEK 293T of the three PHD-insensitive domains of HIF-1α, their stability is compared during normoxia and hypoxia 4 h with PARP activated or inhibited using PJ34 10 μM h PARP-1 domains presenting the full protein, and separately, the DNA-binding, the automodification and the catalytic domain. i pull-Down assay exposing the different domains of PARP-1 to the C-ter domain of HIF-1α. The C-ter binds to PARP-1 full protein and the auto modification domain.

    Article Snippet: In the present work we connect both pathways through HIF-1α C-terminal PARylation via PARP-1.

    Techniques: Construct, Western Blot, Over Expression, Binding Assay, Pull Down Assay, Modification

    Cytotoxic TNF and TRAIL signaling in FADD-RIPK1 and FADD-TRADD double-deficient HeLa-RIPK3 cells. a Western blot evaluation of TRADD, RIPK1, and FADD expression of HeLa-RIPK3 con , HeLa-RIPK3-FADD/RIPK1 DKO and HeLa-RIPK3-FADD/TRADD DKO cells. b Cells were sensitized with 2.5 µg/ml CHX and were stimulated overnight in technical triplicates with the indicated combinations of TNF, TRAIL, ZVAD (20 µM), and nec1 (90 µM). Cellular viability was finally determined by crystal violet staining. A representative panel of experiments is shown. For statistical analysis of independent experiments please see Tables 1 and 2 and Supplementary Tables I – III . c Cells were sensitized with 2.5 µg/ml CHX and stimulated with 100 ng/ml TNF or 100 ng/ml TRAIL for 0–6 h. Total cell lysates were analyzed by western blot for processing of the indicated caspases and caspase substrates. fl full-length

    Journal: Cell Death & Disease

    Article Title: Redundant and receptor-specific activities of TRADD, RIPK1 and FADD in death receptor signaling

    doi: 10.1038/s41419-019-1396-5

    Figure Lengend Snippet: Cytotoxic TNF and TRAIL signaling in FADD-RIPK1 and FADD-TRADD double-deficient HeLa-RIPK3 cells. a Western blot evaluation of TRADD, RIPK1, and FADD expression of HeLa-RIPK3 con , HeLa-RIPK3-FADD/RIPK1 DKO and HeLa-RIPK3-FADD/TRADD DKO cells. b Cells were sensitized with 2.5 µg/ml CHX and were stimulated overnight in technical triplicates with the indicated combinations of TNF, TRAIL, ZVAD (20 µM), and nec1 (90 µM). Cellular viability was finally determined by crystal violet staining. A representative panel of experiments is shown. For statistical analysis of independent experiments please see Tables 1 and 2 and Supplementary Tables I – III . c Cells were sensitized with 2.5 µg/ml CHX and stimulated with 100 ng/ml TNF or 100 ng/ml TRAIL for 0–6 h. Total cell lysates were analyzed by western blot for processing of the indicated caspases and caspase substrates. fl full-length

    Article Snippet: Following primary antibodies have been used: anti-caspase-8 (Santa Cruz, E-20, sc-6133), anti-caspase-8 (Enzo, 5F7), anti-caspase-3 (Cell Signaling, 8G10), anti-caspase-9 (Cell Signaling, # 9502), anti-PARP (BD Biosciences), anti-CYLD (Cell Signaling, D1A10), anti-DR4/TRAILR1 (Cell Signaling, D9S1R), anti-DR5/TRAILR2 (Cell Signaling, D4E9), anti-phospho-RIPK1 (Ser166) (Cell signaling, D1L3S), which is specific for serine 166 phosphorylated necroptosis-competent RIPK1, anti-RIPK1 (Cell Signaling, D94C12), anti-RIPK1 (BD Biosciences, #610459), anti-tubulin (ThermoFisher Scientific (DM1A), anti-TNFR1 (Cell Signaling, C25C1), anti-TRADD (Cell Signaling, 7G8), anti-A20 (Cell Signaling, D13H3), anti-IKKß (Cell Signaling, D30C6), anti-FADD (Cell Signaling, #2782), anti-Sharpin (Abcam ab125188), anti-TRAF2 (Santa Cruz, C-20, sc-876), anti-FLIP (Biomol, NF6, AG-20B-0056), anti-phospho-IκBα (Ser32) (Cell Signaling, 14D4), which recognizes serine 32 phosphorylated IκBα prone for ubiquitination and proteasomal degradation, and anti-IκBα (Cell Signaling, L35A5).

    Techniques: Western Blot, Expressing, Staining

    Evidence for a caspase-8-independent anti-necroptotic activity of FADD in TNFR1 signaling. a HeLa-RIPK3 con , HeLa-RIPK3-FADD KO , and HeLa-RIPK3-casp8 KO cells were analyzed by western blot for expression of caspase-8 and FADD. b Cells were stimulated for the indicated times with 100 ng/ml TNF or 100 ng/ml TRAIL in the presence of 2.5 µg/ml CHX and total cell lysates were analyzed by western blot for processing of the indicated caspases and caspase substrates. fl full-length. c HeLa-RIPK3 variants were stimulated in technical triplicates as indicated with TNF (100 ng/ml), TRAIL (100 ng/ml), CHX (2.5 µg/ml), ZVAD (Z, 20 µM), and nec1 (N, 90 µM). One day later cellular viability was quantified by crystal violet staining. A representative panel of experiments is shown. For statistical analysis of independent experiments please see Tables 1 and 2 and Supplementary Tables I – III . d Cells were treated for the indicated times with TNF (100 ng/ml) or TRAIL (100 ng/ml). Total cell lysates were analyzed by western blot for expression and phosphorylation of RIPK1. e The TRAIL death receptor-associated signaling complex was immunoprecipitated from HeLa-RIPK3 con and HeLa-RIPK3-casp8 KO cells with Fc-TRAIL and protein G beads. IPs were analyzed by western blot for the presence of the indicated proteins. For western blot analysis of lysates see Supplementary Data Fig. S5C

    Journal: Cell Death & Disease

    Article Title: Redundant and receptor-specific activities of TRADD, RIPK1 and FADD in death receptor signaling

    doi: 10.1038/s41419-019-1396-5

    Figure Lengend Snippet: Evidence for a caspase-8-independent anti-necroptotic activity of FADD in TNFR1 signaling. a HeLa-RIPK3 con , HeLa-RIPK3-FADD KO , and HeLa-RIPK3-casp8 KO cells were analyzed by western blot for expression of caspase-8 and FADD. b Cells were stimulated for the indicated times with 100 ng/ml TNF or 100 ng/ml TRAIL in the presence of 2.5 µg/ml CHX and total cell lysates were analyzed by western blot for processing of the indicated caspases and caspase substrates. fl full-length. c HeLa-RIPK3 variants were stimulated in technical triplicates as indicated with TNF (100 ng/ml), TRAIL (100 ng/ml), CHX (2.5 µg/ml), ZVAD (Z, 20 µM), and nec1 (N, 90 µM). One day later cellular viability was quantified by crystal violet staining. A representative panel of experiments is shown. For statistical analysis of independent experiments please see Tables 1 and 2 and Supplementary Tables I – III . d Cells were treated for the indicated times with TNF (100 ng/ml) or TRAIL (100 ng/ml). Total cell lysates were analyzed by western blot for expression and phosphorylation of RIPK1. e The TRAIL death receptor-associated signaling complex was immunoprecipitated from HeLa-RIPK3 con and HeLa-RIPK3-casp8 KO cells with Fc-TRAIL and protein G beads. IPs were analyzed by western blot for the presence of the indicated proteins. For western blot analysis of lysates see Supplementary Data Fig. S5C

    Article Snippet: Following primary antibodies have been used: anti-caspase-8 (Santa Cruz, E-20, sc-6133), anti-caspase-8 (Enzo, 5F7), anti-caspase-3 (Cell Signaling, 8G10), anti-caspase-9 (Cell Signaling, # 9502), anti-PARP (BD Biosciences), anti-CYLD (Cell Signaling, D1A10), anti-DR4/TRAILR1 (Cell Signaling, D9S1R), anti-DR5/TRAILR2 (Cell Signaling, D4E9), anti-phospho-RIPK1 (Ser166) (Cell signaling, D1L3S), which is specific for serine 166 phosphorylated necroptosis-competent RIPK1, anti-RIPK1 (Cell Signaling, D94C12), anti-RIPK1 (BD Biosciences, #610459), anti-tubulin (ThermoFisher Scientific (DM1A), anti-TNFR1 (Cell Signaling, C25C1), anti-TRADD (Cell Signaling, 7G8), anti-A20 (Cell Signaling, D13H3), anti-IKKß (Cell Signaling, D30C6), anti-FADD (Cell Signaling, #2782), anti-Sharpin (Abcam ab125188), anti-TRAF2 (Santa Cruz, C-20, sc-876), anti-FLIP (Biomol, NF6, AG-20B-0056), anti-phospho-IκBα (Ser32) (Cell Signaling, 14D4), which recognizes serine 32 phosphorylated IκBα prone for ubiquitination and proteasomal degradation, and anti-IκBα (Cell Signaling, L35A5).

    Techniques: Activity Assay, Western Blot, Expressing, Staining, Immunoprecipitation

    Model of TNFR1 and TRAIL death receptor signaling. For simplicity, the various literature known modifications (phosphorylation, ubiquitination, processing) and oligomerization events which enable TRADD, FADD, RIPK1 and their binding partners to control the activity of proinflammatory and cytotoxic signaling pathways are not indicated. Please note the dynamics of the cytoplasmic complex is unknown. Thus, it is unclear whether two or more relatively stable complexes are formed that interact secondarily in a transient fashion or whether all proteins can assemble into one type of complex. Double headed arrows refer to protein–protein interactions. Red headed arrows indicate activating/stimulating events. Red dotted blocked lines refer to inhibitory events/effects

    Journal: Cell Death & Disease

    Article Title: Redundant and receptor-specific activities of TRADD, RIPK1 and FADD in death receptor signaling

    doi: 10.1038/s41419-019-1396-5

    Figure Lengend Snippet: Model of TNFR1 and TRAIL death receptor signaling. For simplicity, the various literature known modifications (phosphorylation, ubiquitination, processing) and oligomerization events which enable TRADD, FADD, RIPK1 and their binding partners to control the activity of proinflammatory and cytotoxic signaling pathways are not indicated. Please note the dynamics of the cytoplasmic complex is unknown. Thus, it is unclear whether two or more relatively stable complexes are formed that interact secondarily in a transient fashion or whether all proteins can assemble into one type of complex. Double headed arrows refer to protein–protein interactions. Red headed arrows indicate activating/stimulating events. Red dotted blocked lines refer to inhibitory events/effects

    Article Snippet: Following primary antibodies have been used: anti-caspase-8 (Santa Cruz, E-20, sc-6133), anti-caspase-8 (Enzo, 5F7), anti-caspase-3 (Cell Signaling, 8G10), anti-caspase-9 (Cell Signaling, # 9502), anti-PARP (BD Biosciences), anti-CYLD (Cell Signaling, D1A10), anti-DR4/TRAILR1 (Cell Signaling, D9S1R), anti-DR5/TRAILR2 (Cell Signaling, D4E9), anti-phospho-RIPK1 (Ser166) (Cell signaling, D1L3S), which is specific for serine 166 phosphorylated necroptosis-competent RIPK1, anti-RIPK1 (Cell Signaling, D94C12), anti-RIPK1 (BD Biosciences, #610459), anti-tubulin (ThermoFisher Scientific (DM1A), anti-TNFR1 (Cell Signaling, C25C1), anti-TRADD (Cell Signaling, 7G8), anti-A20 (Cell Signaling, D13H3), anti-IKKß (Cell Signaling, D30C6), anti-FADD (Cell Signaling, #2782), anti-Sharpin (Abcam ab125188), anti-TRAF2 (Santa Cruz, C-20, sc-876), anti-FLIP (Biomol, NF6, AG-20B-0056), anti-phospho-IκBα (Ser32) (Cell Signaling, 14D4), which recognizes serine 32 phosphorylated IκBα prone for ubiquitination and proteasomal degradation, and anti-IκBα (Cell Signaling, L35A5).

    Techniques: Binding Assay, Activity Assay

    TNF and TRAIL induce apoptosis and necroptosis in HeLa-RIPK3 transfectants. a HeLa-EV and HeLa-RIPK3 cells were stimulated overnight with 100 ng/ml TNF or 100 ng/ml TRAIL in the presence and absence of CHX (2.5 µg/ml) and total cell lysates were analyzed by western blot for processing of the indicated caspases and caspase substrates. fl full-length. b Cells were challenged overnight in technical triplicates with the indicated mixtures of TNF (100 ng/ml), TRAIL (100 ng/ml), CHX (2.5 µg/ml), ZVAD (Z, 20 µM), and nec1 (N, 90 µM). Cellular viability was evaluated by crystal violet staining. A representative panel of experiments is shown. For statistical analysis of independent experiments please see Tables 1 and 2 and Supplementary Tables I – III . c HeLa-EV and Hela-RIPK3 cells were treated with the indicated mixtures of 100 ng/ml TNF, 100 ng/ml TRAIL, 2.5 µg/ml CHX (C), and 20 µM ZVAD (Z) for 8 h and RIPK1 phosphorylation was analyzed by western blot. d Hela-RIPK3 CRISPR/Cas9 control cells (HeLa-RIPK3 con cells, see also Fig. 2a ) were treated with the indicated mixtures of TNF (100 ng/ml), TRAIL (100 ng/ml), CHX (2.5 µg/ml), and ZVAD (20 µM) for 0–8 h. Total cell lysates were analyzed for RIPK1 phosphorylation by western blotting

    Journal: Cell Death & Disease

    Article Title: Redundant and receptor-specific activities of TRADD, RIPK1 and FADD in death receptor signaling

    doi: 10.1038/s41419-019-1396-5

    Figure Lengend Snippet: TNF and TRAIL induce apoptosis and necroptosis in HeLa-RIPK3 transfectants. a HeLa-EV and HeLa-RIPK3 cells were stimulated overnight with 100 ng/ml TNF or 100 ng/ml TRAIL in the presence and absence of CHX (2.5 µg/ml) and total cell lysates were analyzed by western blot for processing of the indicated caspases and caspase substrates. fl full-length. b Cells were challenged overnight in technical triplicates with the indicated mixtures of TNF (100 ng/ml), TRAIL (100 ng/ml), CHX (2.5 µg/ml), ZVAD (Z, 20 µM), and nec1 (N, 90 µM). Cellular viability was evaluated by crystal violet staining. A representative panel of experiments is shown. For statistical analysis of independent experiments please see Tables 1 and 2 and Supplementary Tables I – III . c HeLa-EV and Hela-RIPK3 cells were treated with the indicated mixtures of 100 ng/ml TNF, 100 ng/ml TRAIL, 2.5 µg/ml CHX (C), and 20 µM ZVAD (Z) for 8 h and RIPK1 phosphorylation was analyzed by western blot. d Hela-RIPK3 CRISPR/Cas9 control cells (HeLa-RIPK3 con cells, see also Fig. 2a ) were treated with the indicated mixtures of TNF (100 ng/ml), TRAIL (100 ng/ml), CHX (2.5 µg/ml), and ZVAD (20 µM) for 0–8 h. Total cell lysates were analyzed for RIPK1 phosphorylation by western blotting

    Article Snippet: Following primary antibodies have been used: anti-caspase-8 (Santa Cruz, E-20, sc-6133), anti-caspase-8 (Enzo, 5F7), anti-caspase-3 (Cell Signaling, 8G10), anti-caspase-9 (Cell Signaling, # 9502), anti-PARP (BD Biosciences), anti-CYLD (Cell Signaling, D1A10), anti-DR4/TRAILR1 (Cell Signaling, D9S1R), anti-DR5/TRAILR2 (Cell Signaling, D4E9), anti-phospho-RIPK1 (Ser166) (Cell signaling, D1L3S), which is specific for serine 166 phosphorylated necroptosis-competent RIPK1, anti-RIPK1 (Cell Signaling, D94C12), anti-RIPK1 (BD Biosciences, #610459), anti-tubulin (ThermoFisher Scientific (DM1A), anti-TNFR1 (Cell Signaling, C25C1), anti-TRADD (Cell Signaling, 7G8), anti-A20 (Cell Signaling, D13H3), anti-IKKß (Cell Signaling, D30C6), anti-FADD (Cell Signaling, #2782), anti-Sharpin (Abcam ab125188), anti-TRAF2 (Santa Cruz, C-20, sc-876), anti-FLIP (Biomol, NF6, AG-20B-0056), anti-phospho-IκBα (Ser32) (Cell Signaling, 14D4), which recognizes serine 32 phosphorylated IκBα prone for ubiquitination and proteasomal degradation, and anti-IκBα (Cell Signaling, L35A5).

    Techniques: Western Blot, Staining, CRISPR

    Relevance of TRADD, RIPK1, and FADD for caspase activation and cell death induction by TNF and TRAIL. a Western blot evaluation of TRADD, RIPK1, and FADD expression of HeLa-RIPK3 con , HeLa-RIPK3-TRADD KO , HeLa-RIPK3-RIPK1 KO , and HeLa-RIPK3-FADD KO cells. fl full-length. b The various HeLa-RIPK3 variants were stimulated in technical triplicates as indicated with TNF (100 ng/ml), TRAIL (100 ng/ml), CHX (2.5 µg/ml), ZVAD (Z, 20 µM), and nec1 (N, 90 µM). The next day, cellular viability was evaluated by crystal violet staining. A representative panel of experiments is shown. For statistical analysis of independent experiments please see Tables 1 and 2 and Supplementary Tables I – III . c Western blot analysis of phosphorylated RIPK1 in HeLa-RIPK3 and HeLa-RIPK3-FADD KO cells treated for 2, 4, or 8 h with 100 ng/ml of TNF or TRAIL. Where indicated cells were challenged in the presence of CHX (2.5 µg/ml) and ZVAD (20 µM). d TNFR1- and TRAIL death receptor-associated signaling complexes were immunoprecipitated from the various HeLa-RIPK3 variants with a TNFR1-specific Fc fusion protein of TNF or Fc-TRAIL and protein G beads. IPs were analyzed by western blotting for the presence of the indicated proteins. For western blot analysis of lysates see Supplementary Data (Fig. S5A). e HeLa-RIPK3 variants were stimulated overnight in the presence of 2.5 µg/ml CHX with 1, 10, or 100 ng/ml of TNF or TRAIL. Total cell lysates were analyzed by western blot. f HeLa-RIPK3 con and HeLa-RIPK3-TRADD KO cells were challenged in technical triplicates with increasing concentrations of TNF or TRAIL in the presence of the indicated mixtures of CHX (C, 2.5 µg/ml), nec1 (N, 90 µM, apoptotic conditions), and ZVAD (Z, 20 µM, necroptotic conditions). Cellular viability was determined the next day by crystal violet staining

    Journal: Cell Death & Disease

    Article Title: Redundant and receptor-specific activities of TRADD, RIPK1 and FADD in death receptor signaling

    doi: 10.1038/s41419-019-1396-5

    Figure Lengend Snippet: Relevance of TRADD, RIPK1, and FADD for caspase activation and cell death induction by TNF and TRAIL. a Western blot evaluation of TRADD, RIPK1, and FADD expression of HeLa-RIPK3 con , HeLa-RIPK3-TRADD KO , HeLa-RIPK3-RIPK1 KO , and HeLa-RIPK3-FADD KO cells. fl full-length. b The various HeLa-RIPK3 variants were stimulated in technical triplicates as indicated with TNF (100 ng/ml), TRAIL (100 ng/ml), CHX (2.5 µg/ml), ZVAD (Z, 20 µM), and nec1 (N, 90 µM). The next day, cellular viability was evaluated by crystal violet staining. A representative panel of experiments is shown. For statistical analysis of independent experiments please see Tables 1 and 2 and Supplementary Tables I – III . c Western blot analysis of phosphorylated RIPK1 in HeLa-RIPK3 and HeLa-RIPK3-FADD KO cells treated for 2, 4, or 8 h with 100 ng/ml of TNF or TRAIL. Where indicated cells were challenged in the presence of CHX (2.5 µg/ml) and ZVAD (20 µM). d TNFR1- and TRAIL death receptor-associated signaling complexes were immunoprecipitated from the various HeLa-RIPK3 variants with a TNFR1-specific Fc fusion protein of TNF or Fc-TRAIL and protein G beads. IPs were analyzed by western blotting for the presence of the indicated proteins. For western blot analysis of lysates see Supplementary Data (Fig. S5A). e HeLa-RIPK3 variants were stimulated overnight in the presence of 2.5 µg/ml CHX with 1, 10, or 100 ng/ml of TNF or TRAIL. Total cell lysates were analyzed by western blot. f HeLa-RIPK3 con and HeLa-RIPK3-TRADD KO cells were challenged in technical triplicates with increasing concentrations of TNF or TRAIL in the presence of the indicated mixtures of CHX (C, 2.5 µg/ml), nec1 (N, 90 µM, apoptotic conditions), and ZVAD (Z, 20 µM, necroptotic conditions). Cellular viability was determined the next day by crystal violet staining

    Article Snippet: Following primary antibodies have been used: anti-caspase-8 (Santa Cruz, E-20, sc-6133), anti-caspase-8 (Enzo, 5F7), anti-caspase-3 (Cell Signaling, 8G10), anti-caspase-9 (Cell Signaling, # 9502), anti-PARP (BD Biosciences), anti-CYLD (Cell Signaling, D1A10), anti-DR4/TRAILR1 (Cell Signaling, D9S1R), anti-DR5/TRAILR2 (Cell Signaling, D4E9), anti-phospho-RIPK1 (Ser166) (Cell signaling, D1L3S), which is specific for serine 166 phosphorylated necroptosis-competent RIPK1, anti-RIPK1 (Cell Signaling, D94C12), anti-RIPK1 (BD Biosciences, #610459), anti-tubulin (ThermoFisher Scientific (DM1A), anti-TNFR1 (Cell Signaling, C25C1), anti-TRADD (Cell Signaling, 7G8), anti-A20 (Cell Signaling, D13H3), anti-IKKß (Cell Signaling, D30C6), anti-FADD (Cell Signaling, #2782), anti-Sharpin (Abcam ab125188), anti-TRAF2 (Santa Cruz, C-20, sc-876), anti-FLIP (Biomol, NF6, AG-20B-0056), anti-phospho-IκBα (Ser32) (Cell Signaling, 14D4), which recognizes serine 32 phosphorylated IκBα prone for ubiquitination and proteasomal degradation, and anti-IκBα (Cell Signaling, L35A5).

    Techniques: Activation Assay, Western Blot, Expressing, Staining, Immunoprecipitation

    TRADD and RIPK1 act redundantly in proinflammatory death receptor signaling. a Cells were challenged overnight in triplicates with TNF (100 ng/ml) or TRAIL (100 ng/ml) in the absence (upper panel) and presence (lower panel) of a mixture of 20 µM ZVAD and 90 µM necrostatin-1. Next day, supernatants were analyzed for the presence of IL8 by ELISA. Treatment with ZVAD/necrostatin-1 served to prevent effects of cell death on IL8 production (e.g. due to apoptosis-associated activation of caspases). Shown are the results from independent experiments. b Cells were stimulated in the presence of 2.5 µg/ml CHX and 10 µM MLN4924 with 100 ng/ml TNF or 100 ng/ml TRAIL and were analyzed by western blot for expression and phosphorylation of IκBα. MLN4924 has been added to prevent proteasomal degradation of IκBα to avoid underestimation of IκBα phosphorylation. MLN4924 is an inhibitor of the NEDD8-activating enzyme which is required for the functionality of the E3 ligase complex responsible for K48 ubiquitination of IκBα. c TNFR1-associated signaling complexes were immunoprecipitated with a TNFR1-specific Fc-TNF mutant fusion protein and protein G beads. IPs were analyzed by western blot for the presence of the indicated proteins. fl full-length. For western blot analysis of lysates see supplementary Data Fig. S5B. d HeLa-RIPK3 and HeLa-RIPK3-FADD KO cells were stimulated with 100 ng/ml TNF or 100 ng/ml TRAIL overnight. Cells in the lower panel were treated in the presence of 20 µM ZVAD and 90 µM necrostatin-1. Cell supernatants were analyzed for IL8 production. Shown are the results of three independent experiments. ns non-specific; *** p

    Journal: Cell Death & Disease

    Article Title: Redundant and receptor-specific activities of TRADD, RIPK1 and FADD in death receptor signaling

    doi: 10.1038/s41419-019-1396-5

    Figure Lengend Snippet: TRADD and RIPK1 act redundantly in proinflammatory death receptor signaling. a Cells were challenged overnight in triplicates with TNF (100 ng/ml) or TRAIL (100 ng/ml) in the absence (upper panel) and presence (lower panel) of a mixture of 20 µM ZVAD and 90 µM necrostatin-1. Next day, supernatants were analyzed for the presence of IL8 by ELISA. Treatment with ZVAD/necrostatin-1 served to prevent effects of cell death on IL8 production (e.g. due to apoptosis-associated activation of caspases). Shown are the results from independent experiments. b Cells were stimulated in the presence of 2.5 µg/ml CHX and 10 µM MLN4924 with 100 ng/ml TNF or 100 ng/ml TRAIL and were analyzed by western blot for expression and phosphorylation of IκBα. MLN4924 has been added to prevent proteasomal degradation of IκBα to avoid underestimation of IκBα phosphorylation. MLN4924 is an inhibitor of the NEDD8-activating enzyme which is required for the functionality of the E3 ligase complex responsible for K48 ubiquitination of IκBα. c TNFR1-associated signaling complexes were immunoprecipitated with a TNFR1-specific Fc-TNF mutant fusion protein and protein G beads. IPs were analyzed by western blot for the presence of the indicated proteins. fl full-length. For western blot analysis of lysates see supplementary Data Fig. S5B. d HeLa-RIPK3 and HeLa-RIPK3-FADD KO cells were stimulated with 100 ng/ml TNF or 100 ng/ml TRAIL overnight. Cells in the lower panel were treated in the presence of 20 µM ZVAD and 90 µM necrostatin-1. Cell supernatants were analyzed for IL8 production. Shown are the results of three independent experiments. ns non-specific; *** p

    Article Snippet: Following primary antibodies have been used: anti-caspase-8 (Santa Cruz, E-20, sc-6133), anti-caspase-8 (Enzo, 5F7), anti-caspase-3 (Cell Signaling, 8G10), anti-caspase-9 (Cell Signaling, # 9502), anti-PARP (BD Biosciences), anti-CYLD (Cell Signaling, D1A10), anti-DR4/TRAILR1 (Cell Signaling, D9S1R), anti-DR5/TRAILR2 (Cell Signaling, D4E9), anti-phospho-RIPK1 (Ser166) (Cell signaling, D1L3S), which is specific for serine 166 phosphorylated necroptosis-competent RIPK1, anti-RIPK1 (Cell Signaling, D94C12), anti-RIPK1 (BD Biosciences, #610459), anti-tubulin (ThermoFisher Scientific (DM1A), anti-TNFR1 (Cell Signaling, C25C1), anti-TRADD (Cell Signaling, 7G8), anti-A20 (Cell Signaling, D13H3), anti-IKKß (Cell Signaling, D30C6), anti-FADD (Cell Signaling, #2782), anti-Sharpin (Abcam ab125188), anti-TRAF2 (Santa Cruz, C-20, sc-876), anti-FLIP (Biomol, NF6, AG-20B-0056), anti-phospho-IκBα (Ser32) (Cell Signaling, 14D4), which recognizes serine 32 phosphorylated IκBα prone for ubiquitination and proteasomal degradation, and anti-IκBα (Cell Signaling, L35A5).

    Techniques: Activated Clotting Time Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Expressing, Immunoprecipitation, Mutagenesis

    TRADD and RIPK1 act redundantly in apoptotic TNFR1 signaling. a HeLa-RIPK3 con , HeLa-RIPK3-TRADD KO , HeLa-RIPK3-RIPK1 KO , and HeLa-RIPK3-TRADD/RIPK1 DKO cells were evaluated for expression of TRADD, RIPK1, and FADD by western blot. b CHX-sensitized (2.5 µg/ml) cells were challenged overnight in technical triplicates as indicated with TNF (100 ng/ml), TRAIL (100 ng/ml), ZVAD (Z, 20 µM), and nec1 (N, 90 µM) and cellular viability was determined by crystal violet staining. A representative panel of experiments is shown. For statistical analysis of independent experiments please see Tables 1 and 2 and Supplementary Tables I – III . c HeLa-RIPK3 variants were stimulated for the indicated times with 100 ng/ml TNF or 100 ng/ml TRAIL in the presence of 2.5 µg/ml CHX. Total cell lysates were analyzed by western blotting. d The indicated HeLa-RIPK3 variants were stimulated in triplicates with increasing concentrations of TNF or TRAIL in the presence of the indicated combinations of CHX (2.5 µg/ml), ZVAD (Z, 20 µM), and nec1 (N, 90 µM). Next day, cell viability was evaluated by crystal violet

    Journal: Cell Death & Disease

    Article Title: Redundant and receptor-specific activities of TRADD, RIPK1 and FADD in death receptor signaling

    doi: 10.1038/s41419-019-1396-5

    Figure Lengend Snippet: TRADD and RIPK1 act redundantly in apoptotic TNFR1 signaling. a HeLa-RIPK3 con , HeLa-RIPK3-TRADD KO , HeLa-RIPK3-RIPK1 KO , and HeLa-RIPK3-TRADD/RIPK1 DKO cells were evaluated for expression of TRADD, RIPK1, and FADD by western blot. b CHX-sensitized (2.5 µg/ml) cells were challenged overnight in technical triplicates as indicated with TNF (100 ng/ml), TRAIL (100 ng/ml), ZVAD (Z, 20 µM), and nec1 (N, 90 µM) and cellular viability was determined by crystal violet staining. A representative panel of experiments is shown. For statistical analysis of independent experiments please see Tables 1 and 2 and Supplementary Tables I – III . c HeLa-RIPK3 variants were stimulated for the indicated times with 100 ng/ml TNF or 100 ng/ml TRAIL in the presence of 2.5 µg/ml CHX. Total cell lysates were analyzed by western blotting. d The indicated HeLa-RIPK3 variants were stimulated in triplicates with increasing concentrations of TNF or TRAIL in the presence of the indicated combinations of CHX (2.5 µg/ml), ZVAD (Z, 20 µM), and nec1 (N, 90 µM). Next day, cell viability was evaluated by crystal violet

    Article Snippet: Following primary antibodies have been used: anti-caspase-8 (Santa Cruz, E-20, sc-6133), anti-caspase-8 (Enzo, 5F7), anti-caspase-3 (Cell Signaling, 8G10), anti-caspase-9 (Cell Signaling, # 9502), anti-PARP (BD Biosciences), anti-CYLD (Cell Signaling, D1A10), anti-DR4/TRAILR1 (Cell Signaling, D9S1R), anti-DR5/TRAILR2 (Cell Signaling, D4E9), anti-phospho-RIPK1 (Ser166) (Cell signaling, D1L3S), which is specific for serine 166 phosphorylated necroptosis-competent RIPK1, anti-RIPK1 (Cell Signaling, D94C12), anti-RIPK1 (BD Biosciences, #610459), anti-tubulin (ThermoFisher Scientific (DM1A), anti-TNFR1 (Cell Signaling, C25C1), anti-TRADD (Cell Signaling, 7G8), anti-A20 (Cell Signaling, D13H3), anti-IKKß (Cell Signaling, D30C6), anti-FADD (Cell Signaling, #2782), anti-Sharpin (Abcam ab125188), anti-TRAF2 (Santa Cruz, C-20, sc-876), anti-FLIP (Biomol, NF6, AG-20B-0056), anti-phospho-IκBα (Ser32) (Cell Signaling, 14D4), which recognizes serine 32 phosphorylated IκBα prone for ubiquitination and proteasomal degradation, and anti-IκBα (Cell Signaling, L35A5).

    Techniques: Activated Clotting Time Assay, Expressing, Western Blot, Staining