rabbit anti ripk3  (ProSci Incorporated)


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    ProSci Incorporated rabbit anti ripk3
    A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type <t>RIPK3</t> in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.
    Rabbit Anti Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL"

    Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-04740-w

    A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type RIPK3 in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.
    Figure Legend Snippet: A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type RIPK3 in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.

    Techniques Used: Expressing, Flow Cytometry, Western Blot

    A Three different experimental protocols were used to assess the ability of AMG-47a to inhibit cell death caused by the expression and dimerisation of the MLKL (1-180) -gyrase fusion protein. In protocol 1, inhibitors (or DMSO) were added first, followed by doxycycline and coumermycin together, so that the fusion protein could dimerise on expression. In protocol 2, addition of coumermycin was delayed to allow levels of the fusion protein to accumulate before dimerisation was induced. In protocol 3, the addition of inhibitors (or DMSO) was also delayed, so that it was added after the fusion protein had been expressed for 16 h, but before the addition of coumermycin. In all experimental methods, cells were harvested 24 h after the experiment was initiated. B Wild-type U937 cells expressing the MLKL (1-180) -gyrase fusion protein were treated using either protocol 1, 2, or 3, as described above ( A ). At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent three independent experiments; bars indicate the mean and error bars indicate standard error of the mean. Statistics were calculated in GraphPad Prism8, and p values are shown where <0.05. C Wild-type U937 cells with inducible human RIPK3 were treated with increasing doses of AMG-47a in the presence of 40 nM doxycycline, to induce expression, and 5 µM IDN-6556, to block caspase-dependent cell death. After 48 h, cells were harvested and viability assessed using the Cell-Titer Glo 2 (Promega) system. Data represent two independent experiments (four replicates per experiment) and have been normalised against doxycycline plus IDN-6556 (0% viability) and doxycycline plus IDN-6556 plus NSA (100% viability).
    Figure Legend Snippet: A Three different experimental protocols were used to assess the ability of AMG-47a to inhibit cell death caused by the expression and dimerisation of the MLKL (1-180) -gyrase fusion protein. In protocol 1, inhibitors (or DMSO) were added first, followed by doxycycline and coumermycin together, so that the fusion protein could dimerise on expression. In protocol 2, addition of coumermycin was delayed to allow levels of the fusion protein to accumulate before dimerisation was induced. In protocol 3, the addition of inhibitors (or DMSO) was also delayed, so that it was added after the fusion protein had been expressed for 16 h, but before the addition of coumermycin. In all experimental methods, cells were harvested 24 h after the experiment was initiated. B Wild-type U937 cells expressing the MLKL (1-180) -gyrase fusion protein were treated using either protocol 1, 2, or 3, as described above ( A ). At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent three independent experiments; bars indicate the mean and error bars indicate standard error of the mean. Statistics were calculated in GraphPad Prism8, and p values are shown where <0.05. C Wild-type U937 cells with inducible human RIPK3 were treated with increasing doses of AMG-47a in the presence of 40 nM doxycycline, to induce expression, and 5 µM IDN-6556, to block caspase-dependent cell death. After 48 h, cells were harvested and viability assessed using the Cell-Titer Glo 2 (Promega) system. Data represent two independent experiments (four replicates per experiment) and have been normalised against doxycycline plus IDN-6556 (0% viability) and doxycycline plus IDN-6556 plus NSA (100% viability).

    Techniques Used: Expressing, Flow Cytometry, Blocking Assay

    A, B, C U937, HT29 and MDF cell lines were treated with DMSO, AMG-47a (1 µM for U937 and MDF; 2 µM for HT29), TSI, or a combination of AMG-47a and TSI over a time course as indicated in the individual figure panels. At the conclusion of the experiment, cells were harvested and lysed for western blot analysis. All cell lines were assessed for phosphorylation and total protein of the three key necroptosis effector proteins, RIPK1, RIPK3, and MLKL, and total β-actin was used as a loading control. Where multiple bands are present, the specific band of interest is indicated with a red arrow. Data are representative of at least three independent experiments. U937 ( D ) or MDF ( E ) cell lines were treated with AMG-47a at 10 µM, the RIPK1 inhibitors (GSK-481, Nec-1s) or a RIPK3 inhibitor (GSK-872) at 20 µM, or an equivalent amount of DMSO, for 1 h at 37 °C. Cell suspensions were then heated over an increasing temperature gradient (as indicated in the methods) for 3 min. Cells were then lysed, and the amount of remaining soluble protein at each temperature point was analysed using Western blot. Results are representative of two (RIPK1 and RIPK3 in U937) or three (all other analyses) independent experiments. AMG-47a binding to RIPK1 ( F ) and RIPK3 ( G ) was measured in competitive binding assays using the KINOMEscan ® Assay Platform (DiscoverX). Graphs were plotted using the raw data supplied from DiscoverX, and a curve was fitted using non-linear regression (GraphPad Prism) to determine the K d and associated error (95% confidence interval). Data represent one (RIPK3) or two (RIPK1) independent experiments, with two runs performed in all experiments and each colour on the graph representing a separate run. All data points were used for the non-linear regression. K d determinations for individual replicates can be found in supplementary data (Supplementary Fig. ). H , I AMG-47a was serially diluted from a maximum concentration of 50 µM (RIPK1) or 100 µM (RIPK3), and its ability to inhibit human RIPK1 or RIPK3 kinase activity was assessed using the ADP-Glo Kinase Assay (Promega). The reaction was allowed to proceed for 4 h before readouts were performed, and data were normalised to DMSO (0% inhibition) and either 1 µM GSK-481 (100% inhibition of RIPK1) or 1 µM GSK-872 (100% inhibition of RIPK3). Data are a summary of two independent experiments performed in duplicate, and the IC 50 and associated error (95% confidence interval) were determined using non-linear regression, as above. Each colour on the graph represents one independent experiment.
    Figure Legend Snippet: A, B, C U937, HT29 and MDF cell lines were treated with DMSO, AMG-47a (1 µM for U937 and MDF; 2 µM for HT29), TSI, or a combination of AMG-47a and TSI over a time course as indicated in the individual figure panels. At the conclusion of the experiment, cells were harvested and lysed for western blot analysis. All cell lines were assessed for phosphorylation and total protein of the three key necroptosis effector proteins, RIPK1, RIPK3, and MLKL, and total β-actin was used as a loading control. Where multiple bands are present, the specific band of interest is indicated with a red arrow. Data are representative of at least three independent experiments. U937 ( D ) or MDF ( E ) cell lines were treated with AMG-47a at 10 µM, the RIPK1 inhibitors (GSK-481, Nec-1s) or a RIPK3 inhibitor (GSK-872) at 20 µM, or an equivalent amount of DMSO, for 1 h at 37 °C. Cell suspensions were then heated over an increasing temperature gradient (as indicated in the methods) for 3 min. Cells were then lysed, and the amount of remaining soluble protein at each temperature point was analysed using Western blot. Results are representative of two (RIPK1 and RIPK3 in U937) or three (all other analyses) independent experiments. AMG-47a binding to RIPK1 ( F ) and RIPK3 ( G ) was measured in competitive binding assays using the KINOMEscan ® Assay Platform (DiscoverX). Graphs were plotted using the raw data supplied from DiscoverX, and a curve was fitted using non-linear regression (GraphPad Prism) to determine the K d and associated error (95% confidence interval). Data represent one (RIPK3) or two (RIPK1) independent experiments, with two runs performed in all experiments and each colour on the graph representing a separate run. All data points were used for the non-linear regression. K d determinations for individual replicates can be found in supplementary data (Supplementary Fig. ). H , I AMG-47a was serially diluted from a maximum concentration of 50 µM (RIPK1) or 100 µM (RIPK3), and its ability to inhibit human RIPK1 or RIPK3 kinase activity was assessed using the ADP-Glo Kinase Assay (Promega). The reaction was allowed to proceed for 4 h before readouts were performed, and data were normalised to DMSO (0% inhibition) and either 1 µM GSK-481 (100% inhibition of RIPK1) or 1 µM GSK-872 (100% inhibition of RIPK3). Data are a summary of two independent experiments performed in duplicate, and the IC 50 and associated error (95% confidence interval) were determined using non-linear regression, as above. Each colour on the graph represents one independent experiment.

    Techniques Used: Western Blot, Binding Assay, Concentration Assay, Activity Assay, Kinase Assay, Inhibition

    A The cell death induced by forced dimerisation of the human MLKL NTD involves two distinct processes. In the first, MLKL can self-associate into discrete oligomers on addition of doxycycline. In the second, addition of coumermycin promotes the assembly of large multimeric complexes. AMG-47a is unable to inhibit the second process, thus we propose it prevents membrane attachment, translocation, or oligomerisation of the human MLKL NTD through its activity as a kinase inhibitor. As AMG-47a binds RIPK1, and as RIPK1 is required for this form of cell death, we propose the primary target of AMG-47a is RIPK1 in this context. B When a cell receives a necroptotic stimulus, a number of key events occur, beginning with assembly of a necrosome containing RIPK1, RIPK3, and MLKL. This triggers a number of events, beginning with the phosphorylation of MLKL by RIPK3, which enables a conformational change in MLKL that allows oligomerisation, translocation to cellular membranes, and ultimately cell death. Our data suggest an additional role for RIPK1 in human necroptosis by supporting the conformational change, oligomerisation, or membrane translocation of MLKL following phosphorylation by RIPK3.
    Figure Legend Snippet: A The cell death induced by forced dimerisation of the human MLKL NTD involves two distinct processes. In the first, MLKL can self-associate into discrete oligomers on addition of doxycycline. In the second, addition of coumermycin promotes the assembly of large multimeric complexes. AMG-47a is unable to inhibit the second process, thus we propose it prevents membrane attachment, translocation, or oligomerisation of the human MLKL NTD through its activity as a kinase inhibitor. As AMG-47a binds RIPK1, and as RIPK1 is required for this form of cell death, we propose the primary target of AMG-47a is RIPK1 in this context. B When a cell receives a necroptotic stimulus, a number of key events occur, beginning with assembly of a necrosome containing RIPK1, RIPK3, and MLKL. This triggers a number of events, beginning with the phosphorylation of MLKL by RIPK3, which enables a conformational change in MLKL that allows oligomerisation, translocation to cellular membranes, and ultimately cell death. Our data suggest an additional role for RIPK1 in human necroptosis by supporting the conformational change, oligomerisation, or membrane translocation of MLKL following phosphorylation by RIPK3.

    Techniques Used: Translocation Assay, Activity Assay

    rabbit polyclonal anti ripk3  (ProSci Incorporated)


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    ProSci Incorporated rabbit polyclonal anti ripk3
    <t>RIPK3</t> expression levels in baseline biopsies (A–E) Workflow depicting how baseline biopsies were evaluated for RIPK3 scoring and statistical analysis. From a total of 406 available biopsies, 374 were stained and evaluated within this study. 21 biopsies could not be assessed, and 11 biopsies came from transplants that succumbed to surgical complications, leading to their exclusion (B) Representative images of cortical specimens from baseline biopsies. The exact scores of the illustrated specimens with low and high RIPK3 expression are from left to right as follows: 0; 1.0; 2.34 and 3.0. Scale bars as depicted (C) Representative images of negative controls, specifically, (I) tumor-distant non-inflamed and non-fibrotic renal parenchyma from kidneys after tumor nephrectomy; (II) kidneys from end stage allograft failure with severe interstitial fibrosis and tubular atrophy; (III and IV) kidneys with membranous glomerulonephritis and nephrotic proteinuria. Scale bars as depicted (D) Scatterplot (with median reported in red) depicting the distribution of RIPK3 score across the investigated cohort (E) RIPK3 score is significantly higher in biopsies from deceased donors. Data are presented as scatterplot and in the graph the median is reported. p value from Mann-Whitney test is reported in figure.
    Rabbit Polyclonal Anti Ripk3, supplied by ProSci Incorporated, 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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    Images

    1) Product Images from "High RIPK3 expression is associated with a higher risk of early kidney transplant failure"

    Article Title: High RIPK3 expression is associated with a higher risk of early kidney transplant failure

    Journal: iScience

    doi: 10.1016/j.isci.2023.107879

    RIPK3 expression levels in baseline biopsies (A–E) Workflow depicting how baseline biopsies were evaluated for RIPK3 scoring and statistical analysis. From a total of 406 available biopsies, 374 were stained and evaluated within this study. 21 biopsies could not be assessed, and 11 biopsies came from transplants that succumbed to surgical complications, leading to their exclusion (B) Representative images of cortical specimens from baseline biopsies. The exact scores of the illustrated specimens with low and high RIPK3 expression are from left to right as follows: 0; 1.0; 2.34 and 3.0. Scale bars as depicted (C) Representative images of negative controls, specifically, (I) tumor-distant non-inflamed and non-fibrotic renal parenchyma from kidneys after tumor nephrectomy; (II) kidneys from end stage allograft failure with severe interstitial fibrosis and tubular atrophy; (III and IV) kidneys with membranous glomerulonephritis and nephrotic proteinuria. Scale bars as depicted (D) Scatterplot (with median reported in red) depicting the distribution of RIPK3 score across the investigated cohort (E) RIPK3 score is significantly higher in biopsies from deceased donors. Data are presented as scatterplot and in the graph the median is reported. p value from Mann-Whitney test is reported in figure.
    Figure Legend Snippet: RIPK3 expression levels in baseline biopsies (A–E) Workflow depicting how baseline biopsies were evaluated for RIPK3 scoring and statistical analysis. From a total of 406 available biopsies, 374 were stained and evaluated within this study. 21 biopsies could not be assessed, and 11 biopsies came from transplants that succumbed to surgical complications, leading to their exclusion (B) Representative images of cortical specimens from baseline biopsies. The exact scores of the illustrated specimens with low and high RIPK3 expression are from left to right as follows: 0; 1.0; 2.34 and 3.0. Scale bars as depicted (C) Representative images of negative controls, specifically, (I) tumor-distant non-inflamed and non-fibrotic renal parenchyma from kidneys after tumor nephrectomy; (II) kidneys from end stage allograft failure with severe interstitial fibrosis and tubular atrophy; (III and IV) kidneys with membranous glomerulonephritis and nephrotic proteinuria. Scale bars as depicted (D) Scatterplot (with median reported in red) depicting the distribution of RIPK3 score across the investigated cohort (E) RIPK3 score is significantly higher in biopsies from deceased donors. Data are presented as scatterplot and in the graph the median is reported. p value from Mann-Whitney test is reported in figure.

    Techniques Used: Expressing, Staining, MANN-WHITNEY

    Demographic and clinical characteristics of the corresponding donors and recipients of the 374 renal allografts b
    Figure Legend Snippet: Demographic and clinical characteristics of the corresponding donors and recipients of the 374 renal allografts b

    Techniques Used: Transplantation Assay

    RIPK3 expression predicts kidney transplant failure (A) Kaplan-Meier estimates of death-censored transplant failure. Shown are estimates of the probabilities of the primary endpoint (i.e., the permanent need for dialysis after transplantation, which consists of both primary non-function (without surgical complications) and follow up end-stage transplant failure requiring the reinstitution of dialysis) comparing renal allograft baseline biopsies with a RIPK3 score of 0–2.0 (≤2) and greater than 2.0 (>2). Estimates are shown for the first year (left) and for the follow up period from year 2–5 (right). Data were censored for death-censored graft survival at the time of death with a functioning graft, at last day of detected kidney function, and either at 12 months (for one-year transplant failure) or at 60 months (for the follow up period 2–5 years). p-Values were calculated using the log rank test. (B) Kaplan-Meier estimates of non-death-censored transplant failure. Shown are estimates of the probabilities of the secondary endpoint, which was a composite of primary non-function (without surgical complications), follow-up end-stage transplant failure requiring the reinstitution of dialysis, or recipient death with a functioning allograft for renal allograft baseline biopsies, with a RIPK3 score 0 to 2.0 (≤2) and greater than 2.0 (>2). Estimates are shown for first year (left) and for the follow-up period from year 2–5 (right). Data were censored for non-death-censored graft survival at last day of detected kidney function and either at 12 months (for one-year transplant failure) or at 60 months (for the follow-up period 2–5 years. p-values were calculated using the log rank test.
    Figure Legend Snippet: RIPK3 expression predicts kidney transplant failure (A) Kaplan-Meier estimates of death-censored transplant failure. Shown are estimates of the probabilities of the primary endpoint (i.e., the permanent need for dialysis after transplantation, which consists of both primary non-function (without surgical complications) and follow up end-stage transplant failure requiring the reinstitution of dialysis) comparing renal allograft baseline biopsies with a RIPK3 score of 0–2.0 (≤2) and greater than 2.0 (>2). Estimates are shown for the first year (left) and for the follow up period from year 2–5 (right). Data were censored for death-censored graft survival at the time of death with a functioning graft, at last day of detected kidney function, and either at 12 months (for one-year transplant failure) or at 60 months (for the follow up period 2–5 years). p-Values were calculated using the log rank test. (B) Kaplan-Meier estimates of non-death-censored transplant failure. Shown are estimates of the probabilities of the secondary endpoint, which was a composite of primary non-function (without surgical complications), follow-up end-stage transplant failure requiring the reinstitution of dialysis, or recipient death with a functioning allograft for renal allograft baseline biopsies, with a RIPK3 score 0 to 2.0 (≤2) and greater than 2.0 (>2). Estimates are shown for first year (left) and for the follow-up period from year 2–5 (right). Data were censored for non-death-censored graft survival at last day of detected kidney function and either at 12 months (for one-year transplant failure) or at 60 months (for the follow-up period 2–5 years. p-values were calculated using the log rank test.

    Techniques Used: Expressing, Transplantation Assay

    Univariate Cox proportional hazards models for one-year death-censored transplant failure with hazard ratios (HR) and 95% confidence intervals (CI) for known risk factors
    Figure Legend Snippet: Univariate Cox proportional hazards models for one-year death-censored transplant failure with hazard ratios (HR) and 95% confidence intervals (CI) for known risk factors

    Techniques Used: Transplantation Assay

    Multivariate Cox proportional hazards models for one-year death-censored transplant failure with hazard ratios (HR) and 95% confidence intervals (CI) adjusted for donor and recipient associated risk factors
    Figure Legend Snippet: Multivariate Cox proportional hazards models for one-year death-censored transplant failure with hazard ratios (HR) and 95% confidence intervals (CI) adjusted for donor and recipient associated risk factors

    Techniques Used: Transplantation Assay

    Association of the  RIPK3  Score with possible allograft and storage characteristics concerning organ quality
    Figure Legend Snippet: Association of the RIPK3 Score with possible allograft and storage characteristics concerning organ quality

    Techniques Used:

    RIPK3 expression and its association with acute tubular injury (A) Representative images of PAS reaction of cortical specimen with corresponding RIPK3 staining. Scale bar as depicted. (B and C) Frequency distribution of acute tubular injury (ATI) in the whole cohort. p-value from chi-square test is reported in figure (C) Frequency distribution of ATI in living and deceased donation cohorts, stratified above and below the RIPK3 score median. p value from chi-square test is reported in figure.
    Figure Legend Snippet: RIPK3 expression and its association with acute tubular injury (A) Representative images of PAS reaction of cortical specimen with corresponding RIPK3 staining. Scale bar as depicted. (B and C) Frequency distribution of acute tubular injury (ATI) in the whole cohort. p-value from chi-square test is reported in figure (C) Frequency distribution of ATI in living and deceased donation cohorts, stratified above and below the RIPK3 score median. p value from chi-square test is reported in figure.

    Techniques Used: Expressing, Staining


    Figure Legend Snippet:

    Techniques Used: Virus, Recombinant, Saline, Transfection, Purification, Plasmid Preparation, DC Protein Assay, Software, Extraction, Confocal Microscopy

    rabbit anti ripk3  (ProSci Incorporated)


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    ProSci Incorporated rabbit anti ripk3
    ( A ) Schematic of the necroptosis pathway. TNF (T) activates TNFR1, the Smac-mimetic Compound A (S) blocks cIAP activity and the pan-caspase inhibitor Q-VD-OPh (Q) blocks caspase-8 activity. This TSQ stimulus results in activation of RIPK1 and <t>RIPK3,</t> and subsequent phosphorylation and activation of MLKL, which causes MLKL-mediated membrane disruption and cell death. ( B ) Schematic of the constitutively activated mouse MLKL mutant, Q343A. Expression of MLKL Q343A using doxycycline causes cell death in the absence of upstream necroptotic stimuli. This enabled a cell-based phenotypic screen for small molecules that modulate necroptosis at the level or downstream of MLKL activation. The skull and crossbones image (Mycomorphbox_Deadly.png; by Sven Manguard) in ( A , B ) was used under a Creative Commons Attribution-Share Alike 4.0 license. ( C ) Schematic of the cell-based phenotypic screen. A total of 5632 compounds from the WEHI small molecule library along with 40 kinase inhibitors were screened against wild-type or Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the MLKL Q343A mutant. The ability of the small molecules to inhibit cell death was measured by CellTiter-Glo cell viability assays. ABT-869, a VEGF and PDGF receptor tyrosine kinase inhibitor, was identified as a hit. See also Supplementary Figure S1A. ( D ) Chemical structure of ABT-869 and its analogue WEHI-615. ( E ) Wild-type mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( F ) Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( G ) Wild-type mouse dermal fibroblast (MDF) cells were stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) to induce necroptosis and treated with increasing concentrations of ABT-869 or WEHI-615. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 4 independent experiments and errors bars represent SEM.
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    1) Product Images from "The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase"

    Article Title: The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase

    Journal: Biochemical Journal

    doi: 10.1042/BCJ20230035

    ( A ) Schematic of the necroptosis pathway. TNF (T) activates TNFR1, the Smac-mimetic Compound A (S) blocks cIAP activity and the pan-caspase inhibitor Q-VD-OPh (Q) blocks caspase-8 activity. This TSQ stimulus results in activation of RIPK1 and RIPK3, and subsequent phosphorylation and activation of MLKL, which causes MLKL-mediated membrane disruption and cell death. ( B ) Schematic of the constitutively activated mouse MLKL mutant, Q343A. Expression of MLKL Q343A using doxycycline causes cell death in the absence of upstream necroptotic stimuli. This enabled a cell-based phenotypic screen for small molecules that modulate necroptosis at the level or downstream of MLKL activation. The skull and crossbones image (Mycomorphbox_Deadly.png; by Sven Manguard) in ( A , B ) was used under a Creative Commons Attribution-Share Alike 4.0 license. ( C ) Schematic of the cell-based phenotypic screen. A total of 5632 compounds from the WEHI small molecule library along with 40 kinase inhibitors were screened against wild-type or Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the MLKL Q343A mutant. The ability of the small molecules to inhibit cell death was measured by CellTiter-Glo cell viability assays. ABT-869, a VEGF and PDGF receptor tyrosine kinase inhibitor, was identified as a hit. See also Supplementary Figure S1A. ( D ) Chemical structure of ABT-869 and its analogue WEHI-615. ( E ) Wild-type mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( F ) Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( G ) Wild-type mouse dermal fibroblast (MDF) cells were stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) to induce necroptosis and treated with increasing concentrations of ABT-869 or WEHI-615. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 4 independent experiments and errors bars represent SEM.
    Figure Legend Snippet: ( A ) Schematic of the necroptosis pathway. TNF (T) activates TNFR1, the Smac-mimetic Compound A (S) blocks cIAP activity and the pan-caspase inhibitor Q-VD-OPh (Q) blocks caspase-8 activity. This TSQ stimulus results in activation of RIPK1 and RIPK3, and subsequent phosphorylation and activation of MLKL, which causes MLKL-mediated membrane disruption and cell death. ( B ) Schematic of the constitutively activated mouse MLKL mutant, Q343A. Expression of MLKL Q343A using doxycycline causes cell death in the absence of upstream necroptotic stimuli. This enabled a cell-based phenotypic screen for small molecules that modulate necroptosis at the level or downstream of MLKL activation. The skull and crossbones image (Mycomorphbox_Deadly.png; by Sven Manguard) in ( A , B ) was used under a Creative Commons Attribution-Share Alike 4.0 license. ( C ) Schematic of the cell-based phenotypic screen. A total of 5632 compounds from the WEHI small molecule library along with 40 kinase inhibitors were screened against wild-type or Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the MLKL Q343A mutant. The ability of the small molecules to inhibit cell death was measured by CellTiter-Glo cell viability assays. ABT-869, a VEGF and PDGF receptor tyrosine kinase inhibitor, was identified as a hit. See also Supplementary Figure S1A. ( D ) Chemical structure of ABT-869 and its analogue WEHI-615. ( E ) Wild-type mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( F ) Mlkl −/− mouse dermal fibroblast (MDF) cells expressing the doxycycline-inducible MLKL Q343A mutant to trigger constitutive necroptosis were treated with DMSO alone, doxycycline (Dox; 1 µg/ml) and DMSO, or Dox and ABT-869 (1 µM). Cell viability was quantified by CellTiter-Glo. Data represent the mean of ≥2 technical replicates from a single experiment, with individual data points shown. See also Supplementary Figure S1A. ( G ) Wild-type mouse dermal fibroblast (MDF) cells were stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) to induce necroptosis and treated with increasing concentrations of ABT-869 or WEHI-615. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 4 independent experiments and errors bars represent SEM.

    Techniques Used: Activity Assay, Activation Assay, Mutagenesis, Expressing, Staining, Flow Cytometry

    ( A , B ) Wild-type mouse dermal fibroblast (MDF) cells were treated with increasing concentrations of ABT-869 or control compounds, RIPK3 inhibitors GSK′872 and GSK′843, DMSO alone or left untreated (UT) for 1 h then stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) ( A ) or TSZ (TNF, Smac-mimetic, z-VAD-fmk) ( B ) for 24 h to induce necroptosis. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 3 ( A ) or n = 4 ( B ) independent experiments and error bars represent SEM. ( C – F ) Human U937 cells were treated with increasing concentrations of ABT-869 or control compounds, MLKL inhibitor NSA and RIPK1 inhibitor GSK′481, DMSO alone or left untreated (UT) for 1 h then stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) for 48 h ( C ) or TSI (TNF, Smac-mimetic, IDN-6556) for 24 h ( E ) to induce necroptosis. Parallel experiments were performed to assess protection of TSQ ( D ) or TSI ( F ) induced death in the presence of the ABT-869 analogue, WEHI-615. Cell death was monitored by SPY505 (live cells) and propidium iodide (PI; dead cells) uptake using IncuCyte live cell imaging. One representative result shown from n = 4 ( C , D ) or n = 3 ( E , F ) independent experiments. See also Supplementary Figure S2A–H.
    Figure Legend Snippet: ( A , B ) Wild-type mouse dermal fibroblast (MDF) cells were treated with increasing concentrations of ABT-869 or control compounds, RIPK3 inhibitors GSK′872 and GSK′843, DMSO alone or left untreated (UT) for 1 h then stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) ( A ) or TSZ (TNF, Smac-mimetic, z-VAD-fmk) ( B ) for 24 h to induce necroptosis. Cell death was quantified by propidium iodide (PI) staining using flow cytometry. Data represent the mean of n = 3 ( A ) or n = 4 ( B ) independent experiments and error bars represent SEM. ( C – F ) Human U937 cells were treated with increasing concentrations of ABT-869 or control compounds, MLKL inhibitor NSA and RIPK1 inhibitor GSK′481, DMSO alone or left untreated (UT) for 1 h then stimulated with TSQ (TNF, Smac-mimetic, Q-VD-OPh) for 48 h ( C ) or TSI (TNF, Smac-mimetic, IDN-6556) for 24 h ( E ) to induce necroptosis. Parallel experiments were performed to assess protection of TSQ ( D ) or TSI ( F ) induced death in the presence of the ABT-869 analogue, WEHI-615. Cell death was monitored by SPY505 (live cells) and propidium iodide (PI; dead cells) uptake using IncuCyte live cell imaging. One representative result shown from n = 4 ( C , D ) or n = 3 ( E , F ) independent experiments. See also Supplementary Figure S2A–H.

    Techniques Used: Staining, Flow Cytometry, Live Cell Imaging

    ( A ) Binding affinities ( K D ) of ABT-869 and WEHI-615 for human full-length MLKL, RIPK1 kinase domain and RIPK3 kinase domain measured by competition binding assays from the DiscoverX KINOME scan platform using the Kd ELECT service. Each value is the mean of two replicates. ( B – D ) Cellular Thermal Shift Assays (CETSA) in mouse and human cells. Mlkl −/− mouse dermal fibroblast (MDF) cells expressing MLKL Q343A ( B ), wild-type MDF cells ( C ) and human U937 cells ( D ) were treated with DMSO, ABT-869, WEHI-615, RIPK1 inhibitor Nec-1s, RIPK3 inhibitor GSK′872 or human RIPK1 inhibitor GSK′481 (all 20 µM). Cells were subjected to an increasing temperature gradient focused around the melting temperature of the protein of interest. Following the separation of soluble and insoluble proteins, the remaining soluble proteins were detected by Western blot. Red asterisks denote protein standards. One representative result shown from n = 3 ( B , C ) or n = 2–3 ( D ) independent experiments. See also Supplementary Figure S3A–C.
    Figure Legend Snippet: ( A ) Binding affinities ( K D ) of ABT-869 and WEHI-615 for human full-length MLKL, RIPK1 kinase domain and RIPK3 kinase domain measured by competition binding assays from the DiscoverX KINOME scan platform using the Kd ELECT service. Each value is the mean of two replicates. ( B – D ) Cellular Thermal Shift Assays (CETSA) in mouse and human cells. Mlkl −/− mouse dermal fibroblast (MDF) cells expressing MLKL Q343A ( B ), wild-type MDF cells ( C ) and human U937 cells ( D ) were treated with DMSO, ABT-869, WEHI-615, RIPK1 inhibitor Nec-1s, RIPK3 inhibitor GSK′872 or human RIPK1 inhibitor GSK′481 (all 20 µM). Cells were subjected to an increasing temperature gradient focused around the melting temperature of the protein of interest. Following the separation of soluble and insoluble proteins, the remaining soluble proteins were detected by Western blot. Red asterisks denote protein standards. One representative result shown from n = 3 ( B , C ) or n = 2–3 ( D ) independent experiments. See also Supplementary Figure S3A–C.

    Techniques Used: Binding Assay, Expressing, Western Blot

    Thermal Shift Assays (TSA) with mouse and human RIPK1 and RIPK3 kinase domains. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to alter the melting temperature ( T M ) of mouse RIPK1 (9.5 µg) ( A , B ), human RIPK1 (12 µg) ( C , D ), mouse RIPK3 (10 µg) ( E , F ) and human RIPK3 (6.5 µg) ( G , H ) compared with the positive controls Compound 2 for mouse RIPK1, GSK′481 for human RIPK1 and GSK′872 for mouse and human RIPK3 (all 30 µM). Data represent the mean of n = 3 independent experiments and error bars represent SEM. See also Supplementary Figure S4A–H.
    Figure Legend Snippet: Thermal Shift Assays (TSA) with mouse and human RIPK1 and RIPK3 kinase domains. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to alter the melting temperature ( T M ) of mouse RIPK1 (9.5 µg) ( A , B ), human RIPK1 (12 µg) ( C , D ), mouse RIPK3 (10 µg) ( E , F ) and human RIPK3 (6.5 µg) ( G , H ) compared with the positive controls Compound 2 for mouse RIPK1, GSK′481 for human RIPK1 and GSK′872 for mouse and human RIPK3 (all 30 µM). Data represent the mean of n = 3 independent experiments and error bars represent SEM. See also Supplementary Figure S4A–H.

    Techniques Used:

    ( A – H ) In vitro phosphorylation assays with mouse and human RIPK1 and RIPK3 kinase domains measured by ADP-Glo Kinase Assays. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to inhibit the autophosphorylation (IC 50 ) of mouse RIPK1 (200 nM) ( A , B ), human RIPK1 (200 nM) ( C , D ), mouse RIPK3 (10 nM) ( E , F ) and human RIPK3 (10 nM) ( G , H ). Data represent the mean of n = 3 ( A , B , E , F ) or n = 2 ( C , D , G , H ) independent experiments and error bars represent SEM. ( I ) Cellular phosphorylation assays. Wild-type mouse dermal fibroblast (MDF) cells were treated with DMSO, ABT-869, WEHI-615, RIPK1 inhibitor Nec-1s or RIPK3 inhibitor GSK′872 for 2 h then stimulated with TSI (TNF, Smac-mimetic, IDN-6556) for 2 h to induce autophosphorylation of RIPK1 and RIPK3. Ripk1 −/− Mlkl −/− MDF cells and Ripk3 −/− MDF cells were included as controls. Phospho-RIPK1 and phospho-RIPK3 protein levels were detected from whole cell lysates by Western blot. Red asterisks denote protein standards. One representative result shown from n = 3 independent experiments. See also Supplementary Figure S5A–C.
    Figure Legend Snippet: ( A – H ) In vitro phosphorylation assays with mouse and human RIPK1 and RIPK3 kinase domains measured by ADP-Glo Kinase Assays. Increasing concentrations of ABT-869 or WEHI-615 were tested for their ability to inhibit the autophosphorylation (IC 50 ) of mouse RIPK1 (200 nM) ( A , B ), human RIPK1 (200 nM) ( C , D ), mouse RIPK3 (10 nM) ( E , F ) and human RIPK3 (10 nM) ( G , H ). Data represent the mean of n = 3 ( A , B , E , F ) or n = 2 ( C , D , G , H ) independent experiments and error bars represent SEM. ( I ) Cellular phosphorylation assays. Wild-type mouse dermal fibroblast (MDF) cells were treated with DMSO, ABT-869, WEHI-615, RIPK1 inhibitor Nec-1s or RIPK3 inhibitor GSK′872 for 2 h then stimulated with TSI (TNF, Smac-mimetic, IDN-6556) for 2 h to induce autophosphorylation of RIPK1 and RIPK3. Ripk1 −/− Mlkl −/− MDF cells and Ripk3 −/− MDF cells were included as controls. Phospho-RIPK1 and phospho-RIPK3 protein levels were detected from whole cell lysates by Western blot. Red asterisks denote protein standards. One representative result shown from n = 3 independent experiments. See also Supplementary Figure S5A–C.

    Techniques Used: In Vitro, Western Blot


    Figure Legend Snippet:

    Techniques Used: Western Blot, Produced, Transduction


    Figure Legend Snippet:

    Techniques Used:

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    ProSci Incorporated rabbit anti ripk3
    A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type <t>RIPK3</t> in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.
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    1) Product Images from "The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL"

    Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-022-04740-w

    A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type RIPK3 in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.
    Figure Legend Snippet: A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type RIPK3 in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.

    Techniques Used: Expressing, Flow Cytometry, Western Blot

    A Three different experimental protocols were used to assess the ability of AMG-47a to inhibit cell death caused by the expression and dimerisation of the MLKL (1-180) -gyrase fusion protein. In protocol 1, inhibitors (or DMSO) were added first, followed by doxycycline and coumermycin together, so that the fusion protein could dimerise on expression. In protocol 2, addition of coumermycin was delayed to allow levels of the fusion protein to accumulate before dimerisation was induced. In protocol 3, the addition of inhibitors (or DMSO) was also delayed, so that it was added after the fusion protein had been expressed for 16 h, but before the addition of coumermycin. In all experimental methods, cells were harvested 24 h after the experiment was initiated. B Wild-type U937 cells expressing the MLKL (1-180) -gyrase fusion protein were treated using either protocol 1, 2, or 3, as described above ( A ). At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent three independent experiments; bars indicate the mean and error bars indicate standard error of the mean. Statistics were calculated in GraphPad Prism8, and p values are shown where <0.05. C Wild-type U937 cells with inducible human RIPK3 were treated with increasing doses of AMG-47a in the presence of 40 nM doxycycline, to induce expression, and 5 µM IDN-6556, to block caspase-dependent cell death. After 48 h, cells were harvested and viability assessed using the Cell-Titer Glo 2 (Promega) system. Data represent two independent experiments (four replicates per experiment) and have been normalised against doxycycline plus IDN-6556 (0% viability) and doxycycline plus IDN-6556 plus NSA (100% viability).
    Figure Legend Snippet: A Three different experimental protocols were used to assess the ability of AMG-47a to inhibit cell death caused by the expression and dimerisation of the MLKL (1-180) -gyrase fusion protein. In protocol 1, inhibitors (or DMSO) were added first, followed by doxycycline and coumermycin together, so that the fusion protein could dimerise on expression. In protocol 2, addition of coumermycin was delayed to allow levels of the fusion protein to accumulate before dimerisation was induced. In protocol 3, the addition of inhibitors (or DMSO) was also delayed, so that it was added after the fusion protein had been expressed for 16 h, but before the addition of coumermycin. In all experimental methods, cells were harvested 24 h after the experiment was initiated. B Wild-type U937 cells expressing the MLKL (1-180) -gyrase fusion protein were treated using either protocol 1, 2, or 3, as described above ( A ). At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent three independent experiments; bars indicate the mean and error bars indicate standard error of the mean. Statistics were calculated in GraphPad Prism8, and p values are shown where <0.05. C Wild-type U937 cells with inducible human RIPK3 were treated with increasing doses of AMG-47a in the presence of 40 nM doxycycline, to induce expression, and 5 µM IDN-6556, to block caspase-dependent cell death. After 48 h, cells were harvested and viability assessed using the Cell-Titer Glo 2 (Promega) system. Data represent two independent experiments (four replicates per experiment) and have been normalised against doxycycline plus IDN-6556 (0% viability) and doxycycline plus IDN-6556 plus NSA (100% viability).

    Techniques Used: Expressing, Flow Cytometry, Blocking Assay

    A, B, C U937, HT29 and MDF cell lines were treated with DMSO, AMG-47a (1 µM for U937 and MDF; 2 µM for HT29), TSI, or a combination of AMG-47a and TSI over a time course as indicated in the individual figure panels. At the conclusion of the experiment, cells were harvested and lysed for western blot analysis. All cell lines were assessed for phosphorylation and total protein of the three key necroptosis effector proteins, RIPK1, RIPK3, and MLKL, and total β-actin was used as a loading control. Where multiple bands are present, the specific band of interest is indicated with a red arrow. Data are representative of at least three independent experiments. U937 ( D ) or MDF ( E ) cell lines were treated with AMG-47a at 10 µM, the RIPK1 inhibitors (GSK-481, Nec-1s) or a RIPK3 inhibitor (GSK-872) at 20 µM, or an equivalent amount of DMSO, for 1 h at 37 °C. Cell suspensions were then heated over an increasing temperature gradient (as indicated in the methods) for 3 min. Cells were then lysed, and the amount of remaining soluble protein at each temperature point was analysed using Western blot. Results are representative of two (RIPK1 and RIPK3 in U937) or three (all other analyses) independent experiments. AMG-47a binding to RIPK1 ( F ) and RIPK3 ( G ) was measured in competitive binding assays using the KINOMEscan ® Assay Platform (DiscoverX). Graphs were plotted using the raw data supplied from DiscoverX, and a curve was fitted using non-linear regression (GraphPad Prism) to determine the K d and associated error (95% confidence interval). Data represent one (RIPK3) or two (RIPK1) independent experiments, with two runs performed in all experiments and each colour on the graph representing a separate run. All data points were used for the non-linear regression. K d determinations for individual replicates can be found in supplementary data (Supplementary Fig. ). H , I AMG-47a was serially diluted from a maximum concentration of 50 µM (RIPK1) or 100 µM (RIPK3), and its ability to inhibit human RIPK1 or RIPK3 kinase activity was assessed using the ADP-Glo Kinase Assay (Promega). The reaction was allowed to proceed for 4 h before readouts were performed, and data were normalised to DMSO (0% inhibition) and either 1 µM GSK-481 (100% inhibition of RIPK1) or 1 µM GSK-872 (100% inhibition of RIPK3). Data are a summary of two independent experiments performed in duplicate, and the IC 50 and associated error (95% confidence interval) were determined using non-linear regression, as above. Each colour on the graph represents one independent experiment.
    Figure Legend Snippet: A, B, C U937, HT29 and MDF cell lines were treated with DMSO, AMG-47a (1 µM for U937 and MDF; 2 µM for HT29), TSI, or a combination of AMG-47a and TSI over a time course as indicated in the individual figure panels. At the conclusion of the experiment, cells were harvested and lysed for western blot analysis. All cell lines were assessed for phosphorylation and total protein of the three key necroptosis effector proteins, RIPK1, RIPK3, and MLKL, and total β-actin was used as a loading control. Where multiple bands are present, the specific band of interest is indicated with a red arrow. Data are representative of at least three independent experiments. U937 ( D ) or MDF ( E ) cell lines were treated with AMG-47a at 10 µM, the RIPK1 inhibitors (GSK-481, Nec-1s) or a RIPK3 inhibitor (GSK-872) at 20 µM, or an equivalent amount of DMSO, for 1 h at 37 °C. Cell suspensions were then heated over an increasing temperature gradient (as indicated in the methods) for 3 min. Cells were then lysed, and the amount of remaining soluble protein at each temperature point was analysed using Western blot. Results are representative of two (RIPK1 and RIPK3 in U937) or three (all other analyses) independent experiments. AMG-47a binding to RIPK1 ( F ) and RIPK3 ( G ) was measured in competitive binding assays using the KINOMEscan ® Assay Platform (DiscoverX). Graphs were plotted using the raw data supplied from DiscoverX, and a curve was fitted using non-linear regression (GraphPad Prism) to determine the K d and associated error (95% confidence interval). Data represent one (RIPK3) or two (RIPK1) independent experiments, with two runs performed in all experiments and each colour on the graph representing a separate run. All data points were used for the non-linear regression. K d determinations for individual replicates can be found in supplementary data (Supplementary Fig. ). H , I AMG-47a was serially diluted from a maximum concentration of 50 µM (RIPK1) or 100 µM (RIPK3), and its ability to inhibit human RIPK1 or RIPK3 kinase activity was assessed using the ADP-Glo Kinase Assay (Promega). The reaction was allowed to proceed for 4 h before readouts were performed, and data were normalised to DMSO (0% inhibition) and either 1 µM GSK-481 (100% inhibition of RIPK1) or 1 µM GSK-872 (100% inhibition of RIPK3). Data are a summary of two independent experiments performed in duplicate, and the IC 50 and associated error (95% confidence interval) were determined using non-linear regression, as above. Each colour on the graph represents one independent experiment.

    Techniques Used: Western Blot, Binding Assay, Concentration Assay, Activity Assay, Kinase Assay, Inhibition

    A The cell death induced by forced dimerisation of the human MLKL NTD involves two distinct processes. In the first, MLKL can self-associate into discrete oligomers on addition of doxycycline. In the second, addition of coumermycin promotes the assembly of large multimeric complexes. AMG-47a is unable to inhibit the second process, thus we propose it prevents membrane attachment, translocation, or oligomerisation of the human MLKL NTD through its activity as a kinase inhibitor. As AMG-47a binds RIPK1, and as RIPK1 is required for this form of cell death, we propose the primary target of AMG-47a is RIPK1 in this context. B When a cell receives a necroptotic stimulus, a number of key events occur, beginning with assembly of a necrosome containing RIPK1, RIPK3, and MLKL. This triggers a number of events, beginning with the phosphorylation of MLKL by RIPK3, which enables a conformational change in MLKL that allows oligomerisation, translocation to cellular membranes, and ultimately cell death. Our data suggest an additional role for RIPK1 in human necroptosis by supporting the conformational change, oligomerisation, or membrane translocation of MLKL following phosphorylation by RIPK3.
    Figure Legend Snippet: A The cell death induced by forced dimerisation of the human MLKL NTD involves two distinct processes. In the first, MLKL can self-associate into discrete oligomers on addition of doxycycline. In the second, addition of coumermycin promotes the assembly of large multimeric complexes. AMG-47a is unable to inhibit the second process, thus we propose it prevents membrane attachment, translocation, or oligomerisation of the human MLKL NTD through its activity as a kinase inhibitor. As AMG-47a binds RIPK1, and as RIPK1 is required for this form of cell death, we propose the primary target of AMG-47a is RIPK1 in this context. B When a cell receives a necroptotic stimulus, a number of key events occur, beginning with assembly of a necrosome containing RIPK1, RIPK3, and MLKL. This triggers a number of events, beginning with the phosphorylation of MLKL by RIPK3, which enables a conformational change in MLKL that allows oligomerisation, translocation to cellular membranes, and ultimately cell death. Our data suggest an additional role for RIPK1 in human necroptosis by supporting the conformational change, oligomerisation, or membrane translocation of MLKL following phosphorylation by RIPK3.

    Techniques Used: Translocation Assay, Activity Assay

    rabbit anti ripk3  (ProSci Incorporated)


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    ProSci Incorporated rabbit anti ripk3
    RAGE mediates DAMP release following transfusion and RAGE deletion attenuates necroptotic cell death. (a) Plasma HMGB1 and <t>RIPK3</t> following RBC transfusion in WT and RAGE KO mice (n = 3 from 2 independent experiments and * P <0.002, **P = 0.023 for HMGB1; n = 3 from three independent experiments and *P = 0.003, **P = 0.02 for RIPK3). (b) HUVEC were treated with TNFα (100 ng/ml), LPS (10 ng/ml), or CpG (10 μg/ml) in the presence of zVAD for 4 h. RAGE knockdown led to a significant attenuation in cell death following TNFα-zVAD, LPS-zVAD and CpG-zVAD treatment (n = 4 from two independent experiments, P = 0.036 for TNFα, P = 0.035 for LPS, P = 0.005 for CpG). (c) RAGE and RIPK3 expression in naive or TNFα-ZVAD treated HMVEC-L. Proximity Ligation Assay of RAGE and RIPK3 in naive and TNFα-zVAD treated HMVEC-L, RAGE-RIPK3-RED, F-Actin-Green, DAPI-Blue.
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    1) Product Images from "RAGE interacts with the necroptotic protein RIPK3 and mediates transfusion-induced danger signal release"

    Article Title: RAGE interacts with the necroptotic protein RIPK3 and mediates transfusion-induced danger signal release

    Journal: Vox sanguinis

    doi: 10.1111/vox.12946

    RAGE mediates DAMP release following transfusion and RAGE deletion attenuates necroptotic cell death. (a) Plasma HMGB1 and RIPK3 following RBC transfusion in WT and RAGE KO mice (n = 3 from 2 independent experiments and * P <0.002, **P = 0.023 for HMGB1; n = 3 from three independent experiments and *P = 0.003, **P = 0.02 for RIPK3). (b) HUVEC were treated with TNFα (100 ng/ml), LPS (10 ng/ml), or CpG (10 μg/ml) in the presence of zVAD for 4 h. RAGE knockdown led to a significant attenuation in cell death following TNFα-zVAD, LPS-zVAD and CpG-zVAD treatment (n = 4 from two independent experiments, P = 0.036 for TNFα, P = 0.035 for LPS, P = 0.005 for CpG). (c) RAGE and RIPK3 expression in naive or TNFα-ZVAD treated HMVEC-L. Proximity Ligation Assay of RAGE and RIPK3 in naive and TNFα-zVAD treated HMVEC-L, RAGE-RIPK3-RED, F-Actin-Green, DAPI-Blue.
    Figure Legend Snippet: RAGE mediates DAMP release following transfusion and RAGE deletion attenuates necroptotic cell death. (a) Plasma HMGB1 and RIPK3 following RBC transfusion in WT and RAGE KO mice (n = 3 from 2 independent experiments and * P <0.002, **P = 0.023 for HMGB1; n = 3 from three independent experiments and *P = 0.003, **P = 0.02 for RIPK3). (b) HUVEC were treated with TNFα (100 ng/ml), LPS (10 ng/ml), or CpG (10 μg/ml) in the presence of zVAD for 4 h. RAGE knockdown led to a significant attenuation in cell death following TNFα-zVAD, LPS-zVAD and CpG-zVAD treatment (n = 4 from two independent experiments, P = 0.036 for TNFα, P = 0.035 for LPS, P = 0.005 for CpG). (c) RAGE and RIPK3 expression in naive or TNFα-ZVAD treated HMVEC-L. Proximity Ligation Assay of RAGE and RIPK3 in naive and TNFα-zVAD treated HMVEC-L, RAGE-RIPK3-RED, F-Actin-Green, DAPI-Blue.

    Techniques Used: Expressing, Proximity Ligation Assay

    RAGE and RIPK3 are detected in the nucleus of HMVEC-L. (a) HMVEC-L were transfected with GFP-tagged RAGE. Anti-GFP beads were utilized to pulldown RAGE from membrane (M), nuclear (N) or cytosolic fractions (c). Immunoblotting for RAGE and RIPK3 was performed. (b) HMVEC-L were treated with TNFα-zVAD or RBC-zVAD for 4 h. Cell lysates were fractionated and probed for RIPK3 or RAGE. RAGE and RIPK3 were detected in all three compartments. (c) RAGE and RIPK3 expression in naive, RBC-zVAD, or TNFα-ZVAD treated HMVEC-L (n = 6 from two independent experiments, representative micrographs shown).
    Figure Legend Snippet: RAGE and RIPK3 are detected in the nucleus of HMVEC-L. (a) HMVEC-L were transfected with GFP-tagged RAGE. Anti-GFP beads were utilized to pulldown RAGE from membrane (M), nuclear (N) or cytosolic fractions (c). Immunoblotting for RAGE and RIPK3 was performed. (b) HMVEC-L were treated with TNFα-zVAD or RBC-zVAD for 4 h. Cell lysates were fractionated and probed for RIPK3 or RAGE. RAGE and RIPK3 were detected in all three compartments. (c) RAGE and RIPK3 expression in naive, RBC-zVAD, or TNFα-ZVAD treated HMVEC-L (n = 6 from two independent experiments, representative micrographs shown).

    Techniques Used: Transfection, Western Blot, Expressing

    rabbit anti ripk3  (ProSci Incorporated)


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    ProSci Incorporated rabbit anti ripk3
    RAGE mediates DAMP release following transfusion and RAGE deletion attenuates necroptotic cell death. (a) Plasma HMGB1 and <t>RIPK3</t> following RBC transfusion in WT and RAGE KO mice (n = 3 from 2 independent experiments and * P <0.002, **P = 0.023 for HMGB1; n = 3 from three independent experiments and *P = 0.003, **P = 0.02 for RIPK3). (b) HUVEC were treated with TNFα (100 ng/ml), LPS (10 ng/ml), or CpG (10 μg/ml) in the presence of zVAD for 4 h. RAGE knockdown led to a significant attenuation in cell death following TNFα-zVAD, LPS-zVAD and CpG-zVAD treatment (n = 4 from two independent experiments, P = 0.036 for TNFα, P = 0.035 for LPS, P = 0.005 for CpG). (c) RAGE and RIPK3 expression in naive or TNFα-ZVAD treated HMVEC-L. Proximity Ligation Assay of RAGE and RIPK3 in naive and TNFα-zVAD treated HMVEC-L, RAGE-RIPK3-RED, F-Actin-Green, DAPI-Blue.
    Rabbit Anti Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "RAGE interacts with the necroptotic protein RIPK3 and mediates transfusion-induced danger signal release"

    Article Title: RAGE interacts with the necroptotic protein RIPK3 and mediates transfusion-induced danger signal release

    Journal: Vox sanguinis

    doi: 10.1111/vox.12946

    RAGE mediates DAMP release following transfusion and RAGE deletion attenuates necroptotic cell death. (a) Plasma HMGB1 and RIPK3 following RBC transfusion in WT and RAGE KO mice (n = 3 from 2 independent experiments and * P <0.002, **P = 0.023 for HMGB1; n = 3 from three independent experiments and *P = 0.003, **P = 0.02 for RIPK3). (b) HUVEC were treated with TNFα (100 ng/ml), LPS (10 ng/ml), or CpG (10 μg/ml) in the presence of zVAD for 4 h. RAGE knockdown led to a significant attenuation in cell death following TNFα-zVAD, LPS-zVAD and CpG-zVAD treatment (n = 4 from two independent experiments, P = 0.036 for TNFα, P = 0.035 for LPS, P = 0.005 for CpG). (c) RAGE and RIPK3 expression in naive or TNFα-ZVAD treated HMVEC-L. Proximity Ligation Assay of RAGE and RIPK3 in naive and TNFα-zVAD treated HMVEC-L, RAGE-RIPK3-RED, F-Actin-Green, DAPI-Blue.
    Figure Legend Snippet: RAGE mediates DAMP release following transfusion and RAGE deletion attenuates necroptotic cell death. (a) Plasma HMGB1 and RIPK3 following RBC transfusion in WT and RAGE KO mice (n = 3 from 2 independent experiments and * P <0.002, **P = 0.023 for HMGB1; n = 3 from three independent experiments and *P = 0.003, **P = 0.02 for RIPK3). (b) HUVEC were treated with TNFα (100 ng/ml), LPS (10 ng/ml), or CpG (10 μg/ml) in the presence of zVAD for 4 h. RAGE knockdown led to a significant attenuation in cell death following TNFα-zVAD, LPS-zVAD and CpG-zVAD treatment (n = 4 from two independent experiments, P = 0.036 for TNFα, P = 0.035 for LPS, P = 0.005 for CpG). (c) RAGE and RIPK3 expression in naive or TNFα-ZVAD treated HMVEC-L. Proximity Ligation Assay of RAGE and RIPK3 in naive and TNFα-zVAD treated HMVEC-L, RAGE-RIPK3-RED, F-Actin-Green, DAPI-Blue.

    Techniques Used: Expressing, Proximity Ligation Assay

    RAGE and RIPK3 are detected in the nucleus of HMVEC-L. (a) HMVEC-L were transfected with GFP-tagged RAGE. Anti-GFP beads were utilized to pulldown RAGE from membrane (M), nuclear (N) or cytosolic fractions (c). Immunoblotting for RAGE and RIPK3 was performed. (b) HMVEC-L were treated with TNFα-zVAD or RBC-zVAD for 4 h. Cell lysates were fractionated and probed for RIPK3 or RAGE. RAGE and RIPK3 were detected in all three compartments. (c) RAGE and RIPK3 expression in naive, RBC-zVAD, or TNFα-ZVAD treated HMVEC-L (n = 6 from two independent experiments, representative micrographs shown).
    Figure Legend Snippet: RAGE and RIPK3 are detected in the nucleus of HMVEC-L. (a) HMVEC-L were transfected with GFP-tagged RAGE. Anti-GFP beads were utilized to pulldown RAGE from membrane (M), nuclear (N) or cytosolic fractions (c). Immunoblotting for RAGE and RIPK3 was performed. (b) HMVEC-L were treated with TNFα-zVAD or RBC-zVAD for 4 h. Cell lysates were fractionated and probed for RIPK3 or RAGE. RAGE and RIPK3 were detected in all three compartments. (c) RAGE and RIPK3 expression in naive, RBC-zVAD, or TNFα-ZVAD treated HMVEC-L (n = 6 from two independent experiments, representative micrographs shown).

    Techniques Used: Transfection, Western Blot, Expressing

    rabbit polyclonal anti ripk3  (ProSci Incorporated)


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    ProSci Incorporated rabbit polyclonal anti ripk3
    (A) IAV-induced cell death kinetics in primary wild-type (WT), Zbp1−/−, and <t>Ripk3−/−</t> MEFs.
    Rabbit Polyclonal Anti Ripk3, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis"

    Article Title: Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis

    Journal: Cell

    doi: 10.1016/j.cell.2020.02.050

    (A) IAV-induced cell death kinetics in primary wild-type (WT), Zbp1−/−, and Ripk3−/− MEFs.
    Figure Legend Snippet: (A) IAV-induced cell death kinetics in primary wild-type (WT), Zbp1−/−, and Ripk3−/− MEFs.

    Techniques Used:

    (A) IAV-infected (PR8, MOI = 2) WT MEFs were lysed at the indicated times p.i., separated into nuclear and cytoplasmic fractions, and examined for ZBP1, MLKL, RIPK3, and viral proteins by immunoblotting. Immunoblotting for GAPDH and histone H3 was used to confirm purity of cytoplasmic and nuclear fractions.
    Figure Legend Snippet: (A) IAV-infected (PR8, MOI = 2) WT MEFs were lysed at the indicated times p.i., separated into nuclear and cytoplasmic fractions, and examined for ZBP1, MLKL, RIPK3, and viral proteins by immunoblotting. Immunoblotting for GAPDH and histone H3 was used to confirm purity of cytoplasmic and nuclear fractions.

    Techniques Used: Infection, Western Blot

    (A) Survival analysis of age- and sex-matched WT (C57BL/6J) (n = 9), Zbp1−/− (n = 9), Ripk3−/− (n = 9), and Mlkl−/− (n = 8) mice infected with a modestly lethal (~LD20) dose of IAV (PR8, 2,500 EID50/mouse intranasally [i.n.]).
    Figure Legend Snippet: (A) Survival analysis of age- and sex-matched WT (C57BL/6J) (n = 9), Zbp1−/− (n = 9), Ripk3−/− (n = 9), and Mlkl−/− (n = 8) mice infected with a modestly lethal (~LD20) dose of IAV (PR8, 2,500 EID50/mouse intranasally [i.n.]).

    Techniques Used: Infection

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Immunoprecipitation, RNA Sequencing Assay, Plasmid Preparation, Software

    rip3 antibody  (ProSci Incorporated)


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    ProSci Incorporated rip3 antibody
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    ProSci Incorporated rabbit anti ripk3
    A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type <t>RIPK3</t> in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.
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    ProSci Incorporated rabbit polyclonal anti ripk3
    <t>RIPK3</t> expression levels in baseline biopsies (A–E) Workflow depicting how baseline biopsies were evaluated for RIPK3 scoring and statistical analysis. From a total of 406 available biopsies, 374 were stained and evaluated within this study. 21 biopsies could not be assessed, and 11 biopsies came from transplants that succumbed to surgical complications, leading to their exclusion (B) Representative images of cortical specimens from baseline biopsies. The exact scores of the illustrated specimens with low and high RIPK3 expression are from left to right as follows: 0; 1.0; 2.34 and 3.0. Scale bars as depicted (C) Representative images of negative controls, specifically, (I) tumor-distant non-inflamed and non-fibrotic renal parenchyma from kidneys after tumor nephrectomy; (II) kidneys from end stage allograft failure with severe interstitial fibrosis and tubular atrophy; (III and IV) kidneys with membranous glomerulonephritis and nephrotic proteinuria. Scale bars as depicted (D) Scatterplot (with median reported in red) depicting the distribution of RIPK3 score across the investigated cohort (E) RIPK3 score is significantly higher in biopsies from deceased donors. Data are presented as scatterplot and in the graph the median is reported. p value from Mann-Whitney test is reported in figure.
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    <t>RIPK3</t> expression levels in baseline biopsies (A–E) Workflow depicting how baseline biopsies were evaluated for RIPK3 scoring and statistical analysis. From a total of 406 available biopsies, 374 were stained and evaluated within this study. 21 biopsies could not be assessed, and 11 biopsies came from transplants that succumbed to surgical complications, leading to their exclusion (B) Representative images of cortical specimens from baseline biopsies. The exact scores of the illustrated specimens with low and high RIPK3 expression are from left to right as follows: 0; 1.0; 2.34 and 3.0. Scale bars as depicted (C) Representative images of negative controls, specifically, (I) tumor-distant non-inflamed and non-fibrotic renal parenchyma from kidneys after tumor nephrectomy; (II) kidneys from end stage allograft failure with severe interstitial fibrosis and tubular atrophy; (III and IV) kidneys with membranous glomerulonephritis and nephrotic proteinuria. Scale bars as depicted (D) Scatterplot (with median reported in red) depicting the distribution of RIPK3 score across the investigated cohort (E) RIPK3 score is significantly higher in biopsies from deceased donors. Data are presented as scatterplot and in the graph the median is reported. p value from Mann-Whitney test is reported in figure.
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    <t>RIPK3</t> expression levels in baseline biopsies (A–E) Workflow depicting how baseline biopsies were evaluated for RIPK3 scoring and statistical analysis. From a total of 406 available biopsies, 374 were stained and evaluated within this study. 21 biopsies could not be assessed, and 11 biopsies came from transplants that succumbed to surgical complications, leading to their exclusion (B) Representative images of cortical specimens from baseline biopsies. The exact scores of the illustrated specimens with low and high RIPK3 expression are from left to right as follows: 0; 1.0; 2.34 and 3.0. Scale bars as depicted (C) Representative images of negative controls, specifically, (I) tumor-distant non-inflamed and non-fibrotic renal parenchyma from kidneys after tumor nephrectomy; (II) kidneys from end stage allograft failure with severe interstitial fibrosis and tubular atrophy; (III and IV) kidneys with membranous glomerulonephritis and nephrotic proteinuria. Scale bars as depicted (D) Scatterplot (with median reported in red) depicting the distribution of RIPK3 score across the investigated cohort (E) RIPK3 score is significantly higher in biopsies from deceased donors. Data are presented as scatterplot and in the graph the median is reported. p value from Mann-Whitney test is reported in figure.
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    Image Search Results


    A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type RIPK3 in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.

    Journal: Cell Death & Disease

    Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

    doi: 10.1038/s41419-022-04740-w

    Figure Lengend Snippet: A , B U937 cell lines of various genotypes expressing an inducible MLKL(1-180)-gyrase fusion were treated with doxycycline (to induce expression) and/or coumermycin (to induce dimerisation), or left untreated. After 24 h ( A ) or 48 h ( B ), cells were harvested and assessed for cell death (PI uptake measured using flow cytometry), counting a minimum of 5000 cells. Data represent three independent experiments, with the exception of the BAX −/− BAK −/− cell lines, where n = 2. Statistics were calculated in GraphPad Prism 8, and p values are shown where <0.05. C The same U937 cells used in panel A and B were treated with doxycycline alone and left overnight. After approximately 16 h, cells were harvested and lysed for analysis of fusion protein expression via western blot. The upper MLKL band (*) represents endogenous MLKL, and the lower band (arrow) represents the MLKL (1-180) -gyrase fusion protein. D Wild-type U937 were treated with TNF (100 ng/ml), Smac-mimetic (0.5 µM), and caspase inhibitor IDN-6556 (emricasan; 5 µM) (TSI) to induce necroptosis or TNF and Smac-mimetic (TS) alone to induce apoptosis. In parallel, MLKL −/− U937 expressing an inducible MLKL (1-180) -gyrase fusion were treated with doxycycline and coumermycin in the presence or absence of necrosulfonamide (NSA). Images were taken at the specified timepoints using an IncuCyte S3 System to track morphological changes and PI uptake (shown by red). Cells with a necroptotic phenotype (cellular swelling) are indicate with yellow arrows. Three fields were examined per well with representative data shown. E Wild-type U937 cells were treated with doxycycline (20 nM) to induce expression of human wild-type RIPK3 in the presence of various cell death inhibitors, as indicated, for 24 h. At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent four or more independent experiments. F Wild-type, MLKL −/− and RIPK1 −/− U937 expressing inducible human wild-type RIPK3 were treated with doxycycline (20 nM) and IDN-6556 (5 µM), doxycycline alone, or left untreated. After 24 h, cell death was measured as PI uptake using flow cytometry. Data are a summary of six independent experiments.

    Article Snippet: The following primary antibodies were used in this study: rat anti-MLKL clone 3H1 (available as MABC604 from Merck Millipore; 1:1000) was produced in-house as previously described [ ]; rabbit anti-human MLKL pS358 (ab187091, Abcam, Cambridge, United Kingdom; 1:4000); rabbit anti-mouse MLKL pS345 (ab196436, Abcam; 1:1000); rat anti-human RIPK3 (clone 1H2; available as MABC1640 from Merck Millipore; 1:1000) and rat anti-mouse RIPK3 (clone 8G7; available as MABC1595 from Merck Millipore; 1:1000) produced in-house as previously described [ , ]; rabbit anti-RIPK3 (2283, ProSci; 1:1000); rabbit anti-human RIPK3 pS227 (ab209384, Abcam; 1:2000); rabbit anti-mouse RIPK3 pT231/pS232 (GEN135-35-9, kindly supplied by Genentech, South San Francisco, CA, USA [ ]; 1:2000); mouse anti-RIPK1 (610458, BD Transduction Laboratories, San Jose, CA, USA; 1:1000); rabbit anti-RIPK1 (3493, Cell Signalling Technology, Danvers, MA, USA; 1:1000); rabbit anti-human RIPK1 pS166 (65746, Cell Signalling Technology; 1:1000); rabbit anti-mouse RIPK1 pS166 (31122, Cell Signalling Technology; 1:1000); rabbit anti-mouse CASP8 (4927, Cell Signalling Technology; 1:1000); rabbit anti-BAX (2772, Cell Signalling Technology; 1:1000); rabbit anti-BAX (06-536, Upstate Biotechnology, Lake Placid, NY, USA; 1:1000); rabbit anti-GAPDH (2118, Cell Signalling Technology; 1:1000); and mouse anti-β-Actin (A1978, Sigma-Aldrich; 1:3000).

    Techniques: Expressing, Flow Cytometry, Western Blot

    A Three different experimental protocols were used to assess the ability of AMG-47a to inhibit cell death caused by the expression and dimerisation of the MLKL (1-180) -gyrase fusion protein. In protocol 1, inhibitors (or DMSO) were added first, followed by doxycycline and coumermycin together, so that the fusion protein could dimerise on expression. In protocol 2, addition of coumermycin was delayed to allow levels of the fusion protein to accumulate before dimerisation was induced. In protocol 3, the addition of inhibitors (or DMSO) was also delayed, so that it was added after the fusion protein had been expressed for 16 h, but before the addition of coumermycin. In all experimental methods, cells were harvested 24 h after the experiment was initiated. B Wild-type U937 cells expressing the MLKL (1-180) -gyrase fusion protein were treated using either protocol 1, 2, or 3, as described above ( A ). At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent three independent experiments; bars indicate the mean and error bars indicate standard error of the mean. Statistics were calculated in GraphPad Prism8, and p values are shown where <0.05. C Wild-type U937 cells with inducible human RIPK3 were treated with increasing doses of AMG-47a in the presence of 40 nM doxycycline, to induce expression, and 5 µM IDN-6556, to block caspase-dependent cell death. After 48 h, cells were harvested and viability assessed using the Cell-Titer Glo 2 (Promega) system. Data represent two independent experiments (four replicates per experiment) and have been normalised against doxycycline plus IDN-6556 (0% viability) and doxycycline plus IDN-6556 plus NSA (100% viability).

    Journal: Cell Death & Disease

    Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

    doi: 10.1038/s41419-022-04740-w

    Figure Lengend Snippet: A Three different experimental protocols were used to assess the ability of AMG-47a to inhibit cell death caused by the expression and dimerisation of the MLKL (1-180) -gyrase fusion protein. In protocol 1, inhibitors (or DMSO) were added first, followed by doxycycline and coumermycin together, so that the fusion protein could dimerise on expression. In protocol 2, addition of coumermycin was delayed to allow levels of the fusion protein to accumulate before dimerisation was induced. In protocol 3, the addition of inhibitors (or DMSO) was also delayed, so that it was added after the fusion protein had been expressed for 16 h, but before the addition of coumermycin. In all experimental methods, cells were harvested 24 h after the experiment was initiated. B Wild-type U937 cells expressing the MLKL (1-180) -gyrase fusion protein were treated using either protocol 1, 2, or 3, as described above ( A ). At the conclusion of the experiment, cells were analysed for PI uptake using flow cytometry, with a minimum of 5000 cells counted. Data represent three independent experiments; bars indicate the mean and error bars indicate standard error of the mean. Statistics were calculated in GraphPad Prism8, and p values are shown where <0.05. C Wild-type U937 cells with inducible human RIPK3 were treated with increasing doses of AMG-47a in the presence of 40 nM doxycycline, to induce expression, and 5 µM IDN-6556, to block caspase-dependent cell death. After 48 h, cells were harvested and viability assessed using the Cell-Titer Glo 2 (Promega) system. Data represent two independent experiments (four replicates per experiment) and have been normalised against doxycycline plus IDN-6556 (0% viability) and doxycycline plus IDN-6556 plus NSA (100% viability).

    Article Snippet: The following primary antibodies were used in this study: rat anti-MLKL clone 3H1 (available as MABC604 from Merck Millipore; 1:1000) was produced in-house as previously described [ ]; rabbit anti-human MLKL pS358 (ab187091, Abcam, Cambridge, United Kingdom; 1:4000); rabbit anti-mouse MLKL pS345 (ab196436, Abcam; 1:1000); rat anti-human RIPK3 (clone 1H2; available as MABC1640 from Merck Millipore; 1:1000) and rat anti-mouse RIPK3 (clone 8G7; available as MABC1595 from Merck Millipore; 1:1000) produced in-house as previously described [ , ]; rabbit anti-RIPK3 (2283, ProSci; 1:1000); rabbit anti-human RIPK3 pS227 (ab209384, Abcam; 1:2000); rabbit anti-mouse RIPK3 pT231/pS232 (GEN135-35-9, kindly supplied by Genentech, South San Francisco, CA, USA [ ]; 1:2000); mouse anti-RIPK1 (610458, BD Transduction Laboratories, San Jose, CA, USA; 1:1000); rabbit anti-RIPK1 (3493, Cell Signalling Technology, Danvers, MA, USA; 1:1000); rabbit anti-human RIPK1 pS166 (65746, Cell Signalling Technology; 1:1000); rabbit anti-mouse RIPK1 pS166 (31122, Cell Signalling Technology; 1:1000); rabbit anti-mouse CASP8 (4927, Cell Signalling Technology; 1:1000); rabbit anti-BAX (2772, Cell Signalling Technology; 1:1000); rabbit anti-BAX (06-536, Upstate Biotechnology, Lake Placid, NY, USA; 1:1000); rabbit anti-GAPDH (2118, Cell Signalling Technology; 1:1000); and mouse anti-β-Actin (A1978, Sigma-Aldrich; 1:3000).

    Techniques: Expressing, Flow Cytometry, Blocking Assay

    A, B, C U937, HT29 and MDF cell lines were treated with DMSO, AMG-47a (1 µM for U937 and MDF; 2 µM for HT29), TSI, or a combination of AMG-47a and TSI over a time course as indicated in the individual figure panels. At the conclusion of the experiment, cells were harvested and lysed for western blot analysis. All cell lines were assessed for phosphorylation and total protein of the three key necroptosis effector proteins, RIPK1, RIPK3, and MLKL, and total β-actin was used as a loading control. Where multiple bands are present, the specific band of interest is indicated with a red arrow. Data are representative of at least three independent experiments. U937 ( D ) or MDF ( E ) cell lines were treated with AMG-47a at 10 µM, the RIPK1 inhibitors (GSK-481, Nec-1s) or a RIPK3 inhibitor (GSK-872) at 20 µM, or an equivalent amount of DMSO, for 1 h at 37 °C. Cell suspensions were then heated over an increasing temperature gradient (as indicated in the methods) for 3 min. Cells were then lysed, and the amount of remaining soluble protein at each temperature point was analysed using Western blot. Results are representative of two (RIPK1 and RIPK3 in U937) or three (all other analyses) independent experiments. AMG-47a binding to RIPK1 ( F ) and RIPK3 ( G ) was measured in competitive binding assays using the KINOMEscan ® Assay Platform (DiscoverX). Graphs were plotted using the raw data supplied from DiscoverX, and a curve was fitted using non-linear regression (GraphPad Prism) to determine the K d and associated error (95% confidence interval). Data represent one (RIPK3) or two (RIPK1) independent experiments, with two runs performed in all experiments and each colour on the graph representing a separate run. All data points were used for the non-linear regression. K d determinations for individual replicates can be found in supplementary data (Supplementary Fig. ). H , I AMG-47a was serially diluted from a maximum concentration of 50 µM (RIPK1) or 100 µM (RIPK3), and its ability to inhibit human RIPK1 or RIPK3 kinase activity was assessed using the ADP-Glo Kinase Assay (Promega). The reaction was allowed to proceed for 4 h before readouts were performed, and data were normalised to DMSO (0% inhibition) and either 1 µM GSK-481 (100% inhibition of RIPK1) or 1 µM GSK-872 (100% inhibition of RIPK3). Data are a summary of two independent experiments performed in duplicate, and the IC 50 and associated error (95% confidence interval) were determined using non-linear regression, as above. Each colour on the graph represents one independent experiment.

    Journal: Cell Death & Disease

    Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

    doi: 10.1038/s41419-022-04740-w

    Figure Lengend Snippet: A, B, C U937, HT29 and MDF cell lines were treated with DMSO, AMG-47a (1 µM for U937 and MDF; 2 µM for HT29), TSI, or a combination of AMG-47a and TSI over a time course as indicated in the individual figure panels. At the conclusion of the experiment, cells were harvested and lysed for western blot analysis. All cell lines were assessed for phosphorylation and total protein of the three key necroptosis effector proteins, RIPK1, RIPK3, and MLKL, and total β-actin was used as a loading control. Where multiple bands are present, the specific band of interest is indicated with a red arrow. Data are representative of at least three independent experiments. U937 ( D ) or MDF ( E ) cell lines were treated with AMG-47a at 10 µM, the RIPK1 inhibitors (GSK-481, Nec-1s) or a RIPK3 inhibitor (GSK-872) at 20 µM, or an equivalent amount of DMSO, for 1 h at 37 °C. Cell suspensions were then heated over an increasing temperature gradient (as indicated in the methods) for 3 min. Cells were then lysed, and the amount of remaining soluble protein at each temperature point was analysed using Western blot. Results are representative of two (RIPK1 and RIPK3 in U937) or three (all other analyses) independent experiments. AMG-47a binding to RIPK1 ( F ) and RIPK3 ( G ) was measured in competitive binding assays using the KINOMEscan ® Assay Platform (DiscoverX). Graphs were plotted using the raw data supplied from DiscoverX, and a curve was fitted using non-linear regression (GraphPad Prism) to determine the K d and associated error (95% confidence interval). Data represent one (RIPK3) or two (RIPK1) independent experiments, with two runs performed in all experiments and each colour on the graph representing a separate run. All data points were used for the non-linear regression. K d determinations for individual replicates can be found in supplementary data (Supplementary Fig. ). H , I AMG-47a was serially diluted from a maximum concentration of 50 µM (RIPK1) or 100 µM (RIPK3), and its ability to inhibit human RIPK1 or RIPK3 kinase activity was assessed using the ADP-Glo Kinase Assay (Promega). The reaction was allowed to proceed for 4 h before readouts were performed, and data were normalised to DMSO (0% inhibition) and either 1 µM GSK-481 (100% inhibition of RIPK1) or 1 µM GSK-872 (100% inhibition of RIPK3). Data are a summary of two independent experiments performed in duplicate, and the IC 50 and associated error (95% confidence interval) were determined using non-linear regression, as above. Each colour on the graph represents one independent experiment.

    Article Snippet: The following primary antibodies were used in this study: rat anti-MLKL clone 3H1 (available as MABC604 from Merck Millipore; 1:1000) was produced in-house as previously described [ ]; rabbit anti-human MLKL pS358 (ab187091, Abcam, Cambridge, United Kingdom; 1:4000); rabbit anti-mouse MLKL pS345 (ab196436, Abcam; 1:1000); rat anti-human RIPK3 (clone 1H2; available as MABC1640 from Merck Millipore; 1:1000) and rat anti-mouse RIPK3 (clone 8G7; available as MABC1595 from Merck Millipore; 1:1000) produced in-house as previously described [ , ]; rabbit anti-RIPK3 (2283, ProSci; 1:1000); rabbit anti-human RIPK3 pS227 (ab209384, Abcam; 1:2000); rabbit anti-mouse RIPK3 pT231/pS232 (GEN135-35-9, kindly supplied by Genentech, South San Francisco, CA, USA [ ]; 1:2000); mouse anti-RIPK1 (610458, BD Transduction Laboratories, San Jose, CA, USA; 1:1000); rabbit anti-RIPK1 (3493, Cell Signalling Technology, Danvers, MA, USA; 1:1000); rabbit anti-human RIPK1 pS166 (65746, Cell Signalling Technology; 1:1000); rabbit anti-mouse RIPK1 pS166 (31122, Cell Signalling Technology; 1:1000); rabbit anti-mouse CASP8 (4927, Cell Signalling Technology; 1:1000); rabbit anti-BAX (2772, Cell Signalling Technology; 1:1000); rabbit anti-BAX (06-536, Upstate Biotechnology, Lake Placid, NY, USA; 1:1000); rabbit anti-GAPDH (2118, Cell Signalling Technology; 1:1000); and mouse anti-β-Actin (A1978, Sigma-Aldrich; 1:3000).

    Techniques: Western Blot, Binding Assay, Concentration Assay, Activity Assay, Kinase Assay, Inhibition

    A The cell death induced by forced dimerisation of the human MLKL NTD involves two distinct processes. In the first, MLKL can self-associate into discrete oligomers on addition of doxycycline. In the second, addition of coumermycin promotes the assembly of large multimeric complexes. AMG-47a is unable to inhibit the second process, thus we propose it prevents membrane attachment, translocation, or oligomerisation of the human MLKL NTD through its activity as a kinase inhibitor. As AMG-47a binds RIPK1, and as RIPK1 is required for this form of cell death, we propose the primary target of AMG-47a is RIPK1 in this context. B When a cell receives a necroptotic stimulus, a number of key events occur, beginning with assembly of a necrosome containing RIPK1, RIPK3, and MLKL. This triggers a number of events, beginning with the phosphorylation of MLKL by RIPK3, which enables a conformational change in MLKL that allows oligomerisation, translocation to cellular membranes, and ultimately cell death. Our data suggest an additional role for RIPK1 in human necroptosis by supporting the conformational change, oligomerisation, or membrane translocation of MLKL following phosphorylation by RIPK3.

    Journal: Cell Death & Disease

    Article Title: The Lck inhibitor, AMG-47a, blocks necroptosis and implicates RIPK1 in signalling downstream of MLKL

    doi: 10.1038/s41419-022-04740-w

    Figure Lengend Snippet: A The cell death induced by forced dimerisation of the human MLKL NTD involves two distinct processes. In the first, MLKL can self-associate into discrete oligomers on addition of doxycycline. In the second, addition of coumermycin promotes the assembly of large multimeric complexes. AMG-47a is unable to inhibit the second process, thus we propose it prevents membrane attachment, translocation, or oligomerisation of the human MLKL NTD through its activity as a kinase inhibitor. As AMG-47a binds RIPK1, and as RIPK1 is required for this form of cell death, we propose the primary target of AMG-47a is RIPK1 in this context. B When a cell receives a necroptotic stimulus, a number of key events occur, beginning with assembly of a necrosome containing RIPK1, RIPK3, and MLKL. This triggers a number of events, beginning with the phosphorylation of MLKL by RIPK3, which enables a conformational change in MLKL that allows oligomerisation, translocation to cellular membranes, and ultimately cell death. Our data suggest an additional role for RIPK1 in human necroptosis by supporting the conformational change, oligomerisation, or membrane translocation of MLKL following phosphorylation by RIPK3.

    Article Snippet: The following primary antibodies were used in this study: rat anti-MLKL clone 3H1 (available as MABC604 from Merck Millipore; 1:1000) was produced in-house as previously described [ ]; rabbit anti-human MLKL pS358 (ab187091, Abcam, Cambridge, United Kingdom; 1:4000); rabbit anti-mouse MLKL pS345 (ab196436, Abcam; 1:1000); rat anti-human RIPK3 (clone 1H2; available as MABC1640 from Merck Millipore; 1:1000) and rat anti-mouse RIPK3 (clone 8G7; available as MABC1595 from Merck Millipore; 1:1000) produced in-house as previously described [ , ]; rabbit anti-RIPK3 (2283, ProSci; 1:1000); rabbit anti-human RIPK3 pS227 (ab209384, Abcam; 1:2000); rabbit anti-mouse RIPK3 pT231/pS232 (GEN135-35-9, kindly supplied by Genentech, South San Francisco, CA, USA [ ]; 1:2000); mouse anti-RIPK1 (610458, BD Transduction Laboratories, San Jose, CA, USA; 1:1000); rabbit anti-RIPK1 (3493, Cell Signalling Technology, Danvers, MA, USA; 1:1000); rabbit anti-human RIPK1 pS166 (65746, Cell Signalling Technology; 1:1000); rabbit anti-mouse RIPK1 pS166 (31122, Cell Signalling Technology; 1:1000); rabbit anti-mouse CASP8 (4927, Cell Signalling Technology; 1:1000); rabbit anti-BAX (2772, Cell Signalling Technology; 1:1000); rabbit anti-BAX (06-536, Upstate Biotechnology, Lake Placid, NY, USA; 1:1000); rabbit anti-GAPDH (2118, Cell Signalling Technology; 1:1000); and mouse anti-β-Actin (A1978, Sigma-Aldrich; 1:3000).

    Techniques: Translocation Assay, Activity Assay

    RIPK3 expression levels in baseline biopsies (A–E) Workflow depicting how baseline biopsies were evaluated for RIPK3 scoring and statistical analysis. From a total of 406 available biopsies, 374 were stained and evaluated within this study. 21 biopsies could not be assessed, and 11 biopsies came from transplants that succumbed to surgical complications, leading to their exclusion (B) Representative images of cortical specimens from baseline biopsies. The exact scores of the illustrated specimens with low and high RIPK3 expression are from left to right as follows: 0; 1.0; 2.34 and 3.0. Scale bars as depicted (C) Representative images of negative controls, specifically, (I) tumor-distant non-inflamed and non-fibrotic renal parenchyma from kidneys after tumor nephrectomy; (II) kidneys from end stage allograft failure with severe interstitial fibrosis and tubular atrophy; (III and IV) kidneys with membranous glomerulonephritis and nephrotic proteinuria. Scale bars as depicted (D) Scatterplot (with median reported in red) depicting the distribution of RIPK3 score across the investigated cohort (E) RIPK3 score is significantly higher in biopsies from deceased donors. Data are presented as scatterplot and in the graph the median is reported. p value from Mann-Whitney test is reported in figure.

    Journal: iScience

    Article Title: High RIPK3 expression is associated with a higher risk of early kidney transplant failure

    doi: 10.1016/j.isci.2023.107879

    Figure Lengend Snippet: RIPK3 expression levels in baseline biopsies (A–E) Workflow depicting how baseline biopsies were evaluated for RIPK3 scoring and statistical analysis. From a total of 406 available biopsies, 374 were stained and evaluated within this study. 21 biopsies could not be assessed, and 11 biopsies came from transplants that succumbed to surgical complications, leading to their exclusion (B) Representative images of cortical specimens from baseline biopsies. The exact scores of the illustrated specimens with low and high RIPK3 expression are from left to right as follows: 0; 1.0; 2.34 and 3.0. Scale bars as depicted (C) Representative images of negative controls, specifically, (I) tumor-distant non-inflamed and non-fibrotic renal parenchyma from kidneys after tumor nephrectomy; (II) kidneys from end stage allograft failure with severe interstitial fibrosis and tubular atrophy; (III and IV) kidneys with membranous glomerulonephritis and nephrotic proteinuria. Scale bars as depicted (D) Scatterplot (with median reported in red) depicting the distribution of RIPK3 score across the investigated cohort (E) RIPK3 score is significantly higher in biopsies from deceased donors. Data are presented as scatterplot and in the graph the median is reported. p value from Mann-Whitney test is reported in figure.

    Article Snippet: Rabbit polyclonal anti-RIPK3 , ProSci , 2283; RRID: AB_203256.

    Techniques: Expressing, Staining, MANN-WHITNEY

    Demographic and clinical characteristics of the corresponding donors and recipients of the 374 renal allografts b

    Journal: iScience

    Article Title: High RIPK3 expression is associated with a higher risk of early kidney transplant failure

    doi: 10.1016/j.isci.2023.107879

    Figure Lengend Snippet: Demographic and clinical characteristics of the corresponding donors and recipients of the 374 renal allografts b

    Article Snippet: Rabbit polyclonal anti-RIPK3 , ProSci , 2283; RRID: AB_203256.

    Techniques: Transplantation Assay

    RIPK3 expression predicts kidney transplant failure (A) Kaplan-Meier estimates of death-censored transplant failure. Shown are estimates of the probabilities of the primary endpoint (i.e., the permanent need for dialysis after transplantation, which consists of both primary non-function (without surgical complications) and follow up end-stage transplant failure requiring the reinstitution of dialysis) comparing renal allograft baseline biopsies with a RIPK3 score of 0–2.0 (≤2) and greater than 2.0 (>2). Estimates are shown for the first year (left) and for the follow up period from year 2–5 (right). Data were censored for death-censored graft survival at the time of death with a functioning graft, at last day of detected kidney function, and either at 12 months (for one-year transplant failure) or at 60 months (for the follow up period 2–5 years). p-Values were calculated using the log rank test. (B) Kaplan-Meier estimates of non-death-censored transplant failure. Shown are estimates of the probabilities of the secondary endpoint, which was a composite of primary non-function (without surgical complications), follow-up end-stage transplant failure requiring the reinstitution of dialysis, or recipient death with a functioning allograft for renal allograft baseline biopsies, with a RIPK3 score 0 to 2.0 (≤2) and greater than 2.0 (>2). Estimates are shown for first year (left) and for the follow-up period from year 2–5 (right). Data were censored for non-death-censored graft survival at last day of detected kidney function and either at 12 months (for one-year transplant failure) or at 60 months (for the follow-up period 2–5 years. p-values were calculated using the log rank test.

    Journal: iScience

    Article Title: High RIPK3 expression is associated with a higher risk of early kidney transplant failure

    doi: 10.1016/j.isci.2023.107879

    Figure Lengend Snippet: RIPK3 expression predicts kidney transplant failure (A) Kaplan-Meier estimates of death-censored transplant failure. Shown are estimates of the probabilities of the primary endpoint (i.e., the permanent need for dialysis after transplantation, which consists of both primary non-function (without surgical complications) and follow up end-stage transplant failure requiring the reinstitution of dialysis) comparing renal allograft baseline biopsies with a RIPK3 score of 0–2.0 (≤2) and greater than 2.0 (>2). Estimates are shown for the first year (left) and for the follow up period from year 2–5 (right). Data were censored for death-censored graft survival at the time of death with a functioning graft, at last day of detected kidney function, and either at 12 months (for one-year transplant failure) or at 60 months (for the follow up period 2–5 years). p-Values were calculated using the log rank test. (B) Kaplan-Meier estimates of non-death-censored transplant failure. Shown are estimates of the probabilities of the secondary endpoint, which was a composite of primary non-function (without surgical complications), follow-up end-stage transplant failure requiring the reinstitution of dialysis, or recipient death with a functioning allograft for renal allograft baseline biopsies, with a RIPK3 score 0 to 2.0 (≤2) and greater than 2.0 (>2). Estimates are shown for first year (left) and for the follow-up period from year 2–5 (right). Data were censored for non-death-censored graft survival at last day of detected kidney function and either at 12 months (for one-year transplant failure) or at 60 months (for the follow-up period 2–5 years. p-values were calculated using the log rank test.

    Article Snippet: Rabbit polyclonal anti-RIPK3 , ProSci , 2283; RRID: AB_203256.

    Techniques: Expressing, Transplantation Assay

    Univariate Cox proportional hazards models for one-year death-censored transplant failure with hazard ratios (HR) and 95% confidence intervals (CI) for known risk factors

    Journal: iScience

    Article Title: High RIPK3 expression is associated with a higher risk of early kidney transplant failure

    doi: 10.1016/j.isci.2023.107879

    Figure Lengend Snippet: Univariate Cox proportional hazards models for one-year death-censored transplant failure with hazard ratios (HR) and 95% confidence intervals (CI) for known risk factors

    Article Snippet: Rabbit polyclonal anti-RIPK3 , ProSci , 2283; RRID: AB_203256.

    Techniques: Transplantation Assay

    Multivariate Cox proportional hazards models for one-year death-censored transplant failure with hazard ratios (HR) and 95% confidence intervals (CI) adjusted for donor and recipient associated risk factors

    Journal: iScience

    Article Title: High RIPK3 expression is associated with a higher risk of early kidney transplant failure

    doi: 10.1016/j.isci.2023.107879

    Figure Lengend Snippet: Multivariate Cox proportional hazards models for one-year death-censored transplant failure with hazard ratios (HR) and 95% confidence intervals (CI) adjusted for donor and recipient associated risk factors

    Article Snippet: Rabbit polyclonal anti-RIPK3 , ProSci , 2283; RRID: AB_203256.

    Techniques: Transplantation Assay

    Association of the  RIPK3  Score with possible allograft and storage characteristics concerning organ quality

    Journal: iScience

    Article Title: High RIPK3 expression is associated with a higher risk of early kidney transplant failure

    doi: 10.1016/j.isci.2023.107879

    Figure Lengend Snippet: Association of the RIPK3 Score with possible allograft and storage characteristics concerning organ quality

    Article Snippet: Rabbit polyclonal anti-RIPK3 , ProSci , 2283; RRID: AB_203256.

    Techniques:

    RIPK3 expression and its association with acute tubular injury (A) Representative images of PAS reaction of cortical specimen with corresponding RIPK3 staining. Scale bar as depicted. (B and C) Frequency distribution of acute tubular injury (ATI) in the whole cohort. p-value from chi-square test is reported in figure (C) Frequency distribution of ATI in living and deceased donation cohorts, stratified above and below the RIPK3 score median. p value from chi-square test is reported in figure.

    Journal: iScience

    Article Title: High RIPK3 expression is associated with a higher risk of early kidney transplant failure

    doi: 10.1016/j.isci.2023.107879

    Figure Lengend Snippet: RIPK3 expression and its association with acute tubular injury (A) Representative images of PAS reaction of cortical specimen with corresponding RIPK3 staining. Scale bar as depicted. (B and C) Frequency distribution of acute tubular injury (ATI) in the whole cohort. p-value from chi-square test is reported in figure (C) Frequency distribution of ATI in living and deceased donation cohorts, stratified above and below the RIPK3 score median. p value from chi-square test is reported in figure.

    Article Snippet: Rabbit polyclonal anti-RIPK3 , ProSci , 2283; RRID: AB_203256.

    Techniques: Expressing, Staining

    Journal: iScience

    Article Title: High RIPK3 expression is associated with a higher risk of early kidney transplant failure

    doi: 10.1016/j.isci.2023.107879

    Figure Lengend Snippet:

    Article Snippet: Rabbit polyclonal anti-RIPK3 , ProSci , 2283; RRID: AB_203256.

    Techniques: Virus, Recombinant, Saline, Transfection, Purification, Plasmid Preparation, DC Protein Assay, Software, Extraction, Confocal Microscopy