antibodies against ripk3  (ProSci Incorporated)


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    ProSci Incorporated antibodies against ripk3
    Becn1 deficient HSCs show increased GSDME-mediated pyroptosis. (A–C) Representative flow cytometry plots (A) and histograms (B and C) showing cell viability of LSKs from WT and Becn1 vKO mice. Freshly isolated LSKs were cultured for 24 h before cell viability analysis by Annexin V (A and B) or DAPI (C). n = 3 repeats per group, data are shown as mean ± SD. (D) Representative western blot showing the level of <t>RIPK3,</t> MLKL, pMLKL, GSDMD, GSDME, and Caspase-3 (CASP3) in freshly isolated hematopoietic progenitor (c-Kit + ) cells from WT and Becn1 vKO mice. Cell lysates were subjected to immunoblot analysis using indicated antibodies. pMLKL, phosphorylated MLKL; GSDME-FL, full-length GSDME; GSDME-N, the N-terminal product of GSDME. (E) Representative western blot showing the level of RIPK3, MLKL, GSDME, and Caspase-3 in HSCs (CD34 − LSK) from WT and Becn1 vKO mice. Freshly isolated HSCs were cultured for 8 days. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (F) Representative western blot showing the activation of Caspase-3 and GSDME following apoptotic drug treatment. WT c-Kit + cells were treated with ABT263 (10 μM) plus S63845 (10 μM) or mock treated for 5 h. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (G) Representative western blot showing the level of Caspase-3 and GSDME in Caspase-3-shRNA or none target control (NTC) shRNA infected c-Kit + cells with or without apoptosis induction. WT c-Kit + cells were infected with lentivirus carrying an NTC shRNA or a Caspase-3-shRNA for 3 days and then treated with ABT263 (10 μM) plus S63845 (10 μM) or mock treated for 5 h. The infection rate was around 90% and the total cell population was collected for western blot analysis without further isolation. Cell lysates were subjected to immunoblot using indicated antibodies. (H) Representative western blot showing GSDME overexpression in LSKs. Freshly isolated 10 5 LSKs were infected with the full-length GSDME-cDNA or control vector for 3 days. Cell lysates of the total cell population were subjected to western blot using indicated antibodies. (I and J) 40,000 mCherry + cells were isolated from full-length GSDME-cDNA or control vector infected LSKs at 3 days post infection and transplanted into lethally irradiated recipients together with 4.6 × 10 5 competitor cells. Chimera in peripheral blood was evaluated every month until the third month. (I) The schematic diagram showing the experimental design for GSDME (full-length) overexpression transplantation. (J) The line plots depict changes in peripheral blood chimerism of donor-derived cells (CD45.2) in recipients at the indicated time points after transplantation. Data are shown as mean ± SD, n = 5 mice per group. (K) Model for regulation of BECN1 in HSCs cell death.
    Antibodies Against 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
    https://www.bioz.com/result/antibodies against ripk3/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
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    Images

    1) Product Images from "BECN1 modulates hematopoietic stem cells by targeting Caspase-3-GSDME-mediated pyroptosis"

    Article Title: BECN1 modulates hematopoietic stem cells by targeting Caspase-3-GSDME-mediated pyroptosis

    Journal: Blood Science

    doi: 10.1097/BS9.0000000000000051

    Becn1 deficient HSCs show increased GSDME-mediated pyroptosis. (A–C) Representative flow cytometry plots (A) and histograms (B and C) showing cell viability of LSKs from WT and Becn1 vKO mice. Freshly isolated LSKs were cultured for 24 h before cell viability analysis by Annexin V (A and B) or DAPI (C). n = 3 repeats per group, data are shown as mean ± SD. (D) Representative western blot showing the level of RIPK3, MLKL, pMLKL, GSDMD, GSDME, and Caspase-3 (CASP3) in freshly isolated hematopoietic progenitor (c-Kit + ) cells from WT and Becn1 vKO mice. Cell lysates were subjected to immunoblot analysis using indicated antibodies. pMLKL, phosphorylated MLKL; GSDME-FL, full-length GSDME; GSDME-N, the N-terminal product of GSDME. (E) Representative western blot showing the level of RIPK3, MLKL, GSDME, and Caspase-3 in HSCs (CD34 − LSK) from WT and Becn1 vKO mice. Freshly isolated HSCs were cultured for 8 days. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (F) Representative western blot showing the activation of Caspase-3 and GSDME following apoptotic drug treatment. WT c-Kit + cells were treated with ABT263 (10 μM) plus S63845 (10 μM) or mock treated for 5 h. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (G) Representative western blot showing the level of Caspase-3 and GSDME in Caspase-3-shRNA or none target control (NTC) shRNA infected c-Kit + cells with or without apoptosis induction. WT c-Kit + cells were infected with lentivirus carrying an NTC shRNA or a Caspase-3-shRNA for 3 days and then treated with ABT263 (10 μM) plus S63845 (10 μM) or mock treated for 5 h. The infection rate was around 90% and the total cell population was collected for western blot analysis without further isolation. Cell lysates were subjected to immunoblot using indicated antibodies. (H) Representative western blot showing GSDME overexpression in LSKs. Freshly isolated 10 5 LSKs were infected with the full-length GSDME-cDNA or control vector for 3 days. Cell lysates of the total cell population were subjected to western blot using indicated antibodies. (I and J) 40,000 mCherry + cells were isolated from full-length GSDME-cDNA or control vector infected LSKs at 3 days post infection and transplanted into lethally irradiated recipients together with 4.6 × 10 5 competitor cells. Chimera in peripheral blood was evaluated every month until the third month. (I) The schematic diagram showing the experimental design for GSDME (full-length) overexpression transplantation. (J) The line plots depict changes in peripheral blood chimerism of donor-derived cells (CD45.2) in recipients at the indicated time points after transplantation. Data are shown as mean ± SD, n = 5 mice per group. (K) Model for regulation of BECN1 in HSCs cell death.
    Figure Legend Snippet: Becn1 deficient HSCs show increased GSDME-mediated pyroptosis. (A–C) Representative flow cytometry plots (A) and histograms (B and C) showing cell viability of LSKs from WT and Becn1 vKO mice. Freshly isolated LSKs were cultured for 24 h before cell viability analysis by Annexin V (A and B) or DAPI (C). n = 3 repeats per group, data are shown as mean ± SD. (D) Representative western blot showing the level of RIPK3, MLKL, pMLKL, GSDMD, GSDME, and Caspase-3 (CASP3) in freshly isolated hematopoietic progenitor (c-Kit + ) cells from WT and Becn1 vKO mice. Cell lysates were subjected to immunoblot analysis using indicated antibodies. pMLKL, phosphorylated MLKL; GSDME-FL, full-length GSDME; GSDME-N, the N-terminal product of GSDME. (E) Representative western blot showing the level of RIPK3, MLKL, GSDME, and Caspase-3 in HSCs (CD34 − LSK) from WT and Becn1 vKO mice. Freshly isolated HSCs were cultured for 8 days. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (F) Representative western blot showing the activation of Caspase-3 and GSDME following apoptotic drug treatment. WT c-Kit + cells were treated with ABT263 (10 μM) plus S63845 (10 μM) or mock treated for 5 h. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (G) Representative western blot showing the level of Caspase-3 and GSDME in Caspase-3-shRNA or none target control (NTC) shRNA infected c-Kit + cells with or without apoptosis induction. WT c-Kit + cells were infected with lentivirus carrying an NTC shRNA or a Caspase-3-shRNA for 3 days and then treated with ABT263 (10 μM) plus S63845 (10 μM) or mock treated for 5 h. The infection rate was around 90% and the total cell population was collected for western blot analysis without further isolation. Cell lysates were subjected to immunoblot using indicated antibodies. (H) Representative western blot showing GSDME overexpression in LSKs. Freshly isolated 10 5 LSKs were infected with the full-length GSDME-cDNA or control vector for 3 days. Cell lysates of the total cell population were subjected to western blot using indicated antibodies. (I and J) 40,000 mCherry + cells were isolated from full-length GSDME-cDNA or control vector infected LSKs at 3 days post infection and transplanted into lethally irradiated recipients together with 4.6 × 10 5 competitor cells. Chimera in peripheral blood was evaluated every month until the third month. (I) The schematic diagram showing the experimental design for GSDME (full-length) overexpression transplantation. (J) The line plots depict changes in peripheral blood chimerism of donor-derived cells (CD45.2) in recipients at the indicated time points after transplantation. Data are shown as mean ± SD, n = 5 mice per group. (K) Model for regulation of BECN1 in HSCs cell death.

    Techniques Used: Flow Cytometry, Isolation, Cell Culture, Western Blot, Activation Assay, shRNA, Infection, Over Expression, Plasmid Preparation, Irradiation, Transplantation Assay, Derivative Assay

    antibody against ripk3  (ProSci Incorporated)


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    ProSci Incorporated antibody against 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.
    Antibody Against 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
    https://www.bioz.com/result/antibody against ripk3/product/ProSci Incorporated
    Average 86 stars, based on 1 article reviews
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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

    antibodies against ripk3  (ProSci Incorporated)


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    ProSci Incorporated antibodies against ripk3
    Becn1 deficient HSCs show increased GSDME-mediated pyroptosis. (A–C) Representative flow cytometry plots (A) and histograms (B and C) showing cell viability of LSKs from WT and Becn1 vKO mice. Freshly isolated LSKs were cultured for 24 h before cell viability analysis by Annexin V (A and B) or DAPI (C). n = 3 repeats per group, data are shown as mean ± SD. (D) Representative western blot showing the level of <t>RIPK3,</t> MLKL, pMLKL, GSDMD, GSDME, and Caspase-3 (CASP3) in freshly isolated hematopoietic progenitor (c-Kit + ) cells from WT and Becn1 vKO mice. Cell lysates were subjected to immunoblot analysis using indicated antibodies. pMLKL, phosphorylated MLKL; GSDME-FL, full-length GSDME; GSDME-N, the N-terminal product of GSDME. (E) Representative western blot showing the level of RIPK3, MLKL, GSDME, and Caspase-3 in HSCs (CD34 − LSK) from WT and Becn1 vKO mice. Freshly isolated HSCs were cultured for 8 days. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (F) Representative western blot showing the activation of Caspase-3 and GSDME following apoptotic drug treatment. WT c-Kit + cells were treated with ABT263 (10 μM) plus S63845 (10 μM) or mock treated for 5 h. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (G) Representative western blot showing the level of Caspase-3 and GSDME in Caspase-3-shRNA or none target control (NTC) shRNA infected c-Kit + cells with or without apoptosis induction. WT c-Kit + cells were infected with lentivirus carrying an NTC shRNA or a Caspase-3-shRNA for 3 days and then treated with ABT263 (10 μM) plus S63845 (10 μM) or mock treated for 5 h. The infection rate was around 90% and the total cell population was collected for western blot analysis without further isolation. Cell lysates were subjected to immunoblot using indicated antibodies. (H) Representative western blot showing GSDME overexpression in LSKs. Freshly isolated 10 5 LSKs were infected with the full-length GSDME-cDNA or control vector for 3 days. Cell lysates of the total cell population were subjected to western blot using indicated antibodies. (I and J) 40,000 mCherry + cells were isolated from full-length GSDME-cDNA or control vector infected LSKs at 3 days post infection and transplanted into lethally irradiated recipients together with 4.6 × 10 5 competitor cells. Chimera in peripheral blood was evaluated every month until the third month. (I) The schematic diagram showing the experimental design for GSDME (full-length) overexpression transplantation. (J) The line plots depict changes in peripheral blood chimerism of donor-derived cells (CD45.2) in recipients at the indicated time points after transplantation. Data are shown as mean ± SD, n = 5 mice per group. (K) Model for regulation of BECN1 in HSCs cell death.
    Antibodies Against 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
    https://www.bioz.com/result/antibodies against ripk3/product/ProSci Incorporated
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antibodies against ripk3 - by Bioz Stars, 2024-06
    90/100 stars

    Images

    1) Product Images from "BECN1 modulates hematopoietic stem cells by targeting Caspase-3-GSDME-mediated pyroptosis"

    Article Title: BECN1 modulates hematopoietic stem cells by targeting Caspase-3-GSDME-mediated pyroptosis

    Journal: Blood Science

    doi: 10.1097/BS9.0000000000000051

    Becn1 deficient HSCs show increased GSDME-mediated pyroptosis. (A–C) Representative flow cytometry plots (A) and histograms (B and C) showing cell viability of LSKs from WT and Becn1 vKO mice. Freshly isolated LSKs were cultured for 24 h before cell viability analysis by Annexin V (A and B) or DAPI (C). n = 3 repeats per group, data are shown as mean ± SD. (D) Representative western blot showing the level of RIPK3, MLKL, pMLKL, GSDMD, GSDME, and Caspase-3 (CASP3) in freshly isolated hematopoietic progenitor (c-Kit + ) cells from WT and Becn1 vKO mice. Cell lysates were subjected to immunoblot analysis using indicated antibodies. pMLKL, phosphorylated MLKL; GSDME-FL, full-length GSDME; GSDME-N, the N-terminal product of GSDME. (E) Representative western blot showing the level of RIPK3, MLKL, GSDME, and Caspase-3 in HSCs (CD34 − LSK) from WT and Becn1 vKO mice. Freshly isolated HSCs were cultured for 8 days. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (F) Representative western blot showing the activation of Caspase-3 and GSDME following apoptotic drug treatment. WT c-Kit + cells were treated with ABT263 (10 μM) plus S63845 (10 μM) or mock treated for 5 h. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (G) Representative western blot showing the level of Caspase-3 and GSDME in Caspase-3-shRNA or none target control (NTC) shRNA infected c-Kit + cells with or without apoptosis induction. WT c-Kit + cells were infected with lentivirus carrying an NTC shRNA or a Caspase-3-shRNA for 3 days and then treated with ABT263 (10 μM) plus S63845 (10 μM) or mock treated for 5 h. The infection rate was around 90% and the total cell population was collected for western blot analysis without further isolation. Cell lysates were subjected to immunoblot using indicated antibodies. (H) Representative western blot showing GSDME overexpression in LSKs. Freshly isolated 10 5 LSKs were infected with the full-length GSDME-cDNA or control vector for 3 days. Cell lysates of the total cell population were subjected to western blot using indicated antibodies. (I and J) 40,000 mCherry + cells were isolated from full-length GSDME-cDNA or control vector infected LSKs at 3 days post infection and transplanted into lethally irradiated recipients together with 4.6 × 10 5 competitor cells. Chimera in peripheral blood was evaluated every month until the third month. (I) The schematic diagram showing the experimental design for GSDME (full-length) overexpression transplantation. (J) The line plots depict changes in peripheral blood chimerism of donor-derived cells (CD45.2) in recipients at the indicated time points after transplantation. Data are shown as mean ± SD, n = 5 mice per group. (K) Model for regulation of BECN1 in HSCs cell death.
    Figure Legend Snippet: Becn1 deficient HSCs show increased GSDME-mediated pyroptosis. (A–C) Representative flow cytometry plots (A) and histograms (B and C) showing cell viability of LSKs from WT and Becn1 vKO mice. Freshly isolated LSKs were cultured for 24 h before cell viability analysis by Annexin V (A and B) or DAPI (C). n = 3 repeats per group, data are shown as mean ± SD. (D) Representative western blot showing the level of RIPK3, MLKL, pMLKL, GSDMD, GSDME, and Caspase-3 (CASP3) in freshly isolated hematopoietic progenitor (c-Kit + ) cells from WT and Becn1 vKO mice. Cell lysates were subjected to immunoblot analysis using indicated antibodies. pMLKL, phosphorylated MLKL; GSDME-FL, full-length GSDME; GSDME-N, the N-terminal product of GSDME. (E) Representative western blot showing the level of RIPK3, MLKL, GSDME, and Caspase-3 in HSCs (CD34 − LSK) from WT and Becn1 vKO mice. Freshly isolated HSCs were cultured for 8 days. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (F) Representative western blot showing the activation of Caspase-3 and GSDME following apoptotic drug treatment. WT c-Kit + cells were treated with ABT263 (10 μM) plus S63845 (10 μM) or mock treated for 5 h. Cell lysates were subjected to immunoblot analysis using indicated antibodies. (G) Representative western blot showing the level of Caspase-3 and GSDME in Caspase-3-shRNA or none target control (NTC) shRNA infected c-Kit + cells with or without apoptosis induction. WT c-Kit + cells were infected with lentivirus carrying an NTC shRNA or a Caspase-3-shRNA for 3 days and then treated with ABT263 (10 μM) plus S63845 (10 μM) or mock treated for 5 h. The infection rate was around 90% and the total cell population was collected for western blot analysis without further isolation. Cell lysates were subjected to immunoblot using indicated antibodies. (H) Representative western blot showing GSDME overexpression in LSKs. Freshly isolated 10 5 LSKs were infected with the full-length GSDME-cDNA or control vector for 3 days. Cell lysates of the total cell population were subjected to western blot using indicated antibodies. (I and J) 40,000 mCherry + cells were isolated from full-length GSDME-cDNA or control vector infected LSKs at 3 days post infection and transplanted into lethally irradiated recipients together with 4.6 × 10 5 competitor cells. Chimera in peripheral blood was evaluated every month until the third month. (I) The schematic diagram showing the experimental design for GSDME (full-length) overexpression transplantation. (J) The line plots depict changes in peripheral blood chimerism of donor-derived cells (CD45.2) in recipients at the indicated time points after transplantation. Data are shown as mean ± SD, n = 5 mice per group. (K) Model for regulation of BECN1 in HSCs cell death.

    Techniques Used: Flow Cytometry, Isolation, Cell Culture, Western Blot, Activation Assay, shRNA, Infection, Over Expression, Plasmid Preparation, Irradiation, Transplantation Assay, Derivative Assay

    antibodies against ripk3  (ProSci Incorporated)


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    ProSci Incorporated antibodies against ripk3
    Antibodies Against 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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    antibodies against ripk3  (ProSci Incorporated)


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    ProSci Incorporated antibodies against ripk3
    Antibodies Against 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
    https://www.bioz.com/result/antibodies against ripk3/product/ProSci Incorporated
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    antibody against ripk3  (ProSci Incorporated)


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    Structured Review

    ProSci Incorporated antibody against ripk3
    ( A, B ) Macroscopic features and weights of Ripk 3 +/+ (wild-type) and Ripk 3 -/- ( <t>Ripk3</t> -knockout) male mice. Ripk 3 +/+ (4 Month, n = 16; 18 Month, n = 27) and Ripk 3 -/- (4 Month, n = 16; 18 Month, n = 27) male mice were photographed and weighed. Data represent the mean ±the standard error of the mean (s.e.m). **p<0.01, ***p<0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( C, D ) Macroscopic features and weights of seminal vesicles. Mice were sacrificed at 18 months of age, and the seminal vesicles from Ripk 3 +/+ (n = 33) and Ripk 3 -/- (n = 30) mice were photographed and weighed. Data represent the mean ±s.e.m. ***p < 0.001. p values were determined with unpaired Student’s t -tests. ( E ) Serum testosterone levels of mice assayed using ELISA. Mice were sacrificed, and the testosterone levels in serum from Ripk 3 +/+ (4 Month, n = 9; 18 Month, n = 9) and Ripk 3 -/- (4 Month, n = 9; 18 Month, n = 9) mice were measured using an ELISA kit for testosterone. Data represent the mean ±s.e.m. *p<0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( F, G ) H and E of testis sections from Ripk 3 +/+ and Ripk 3 -/- mice. Ripk 3 +/+ (4 Months, n = 10; 18 Months, n = 10) and Ripk 3 -/- (4 Months, n = 10; 18 Months, n = 10) mice were sacrificed and testes were harvested and stained with H and E in ( F ). The number of empty seminiferous tubules was counted based on H and E staining and quantification in ( G ), empty seminiferous tubules were counted in five fields per testis. Scale bar, 100 μm. Data represent the mean ± S.D. ***p < 0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( H ) Summary of the fertility rates of Ripk 3 +/+ and Ripk 3 -/- mice. One male mice of a given age was mated with a pairs of 10-week-old wild-type female mice for 3 months; females were replaced every 2 weeks. The number of male mice with reproductive capacity was counted (see Materials and methods). p values were determined using chi-square tests. ( I ) Reproductive longevity. When Ripk 3 +/+ (n = 12) and Ripk 3 -/- (n = 12) male mice were 2 months old, they were continuously mated with a pairs of 10-week-old female mice until pregnancies ceased; females were replaced every 2 months. The ages of the males at which their last litter was sired was recorded (calculated as the age at birth of the litter less 21 days, see Materials and methods). Data represent the mean ± S.D. **p < 0.01. p values were determined with unpaired Student’s t -tests. DOI: http://dx.doi.org/10.7554/eLife.27692.002 10.7554/eLife.27692.003 Figure 1—source data 1. Summary of the fertility rates and mortality rates of the offspring of 4- or 18-month-old Ripk 3 +/+ and Ripk 3 -/- male mice. DOI: http://dx.doi.org/10.7554/eLife.27692.003 10.7554/eLife.27692.004 Figure 1—source data 2. Summary of the fertility rates and mortality rates of the offspring of 13-month-old Ripk 3 +/+ and Ripk 3 -/- male mice. DOI: http://dx.doi.org/10.7554/eLife.27692.004
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    1) Product Images from "RIPK1-RIPK3-MLKL-dependent necrosis promotes the aging of mouse male reproductive system"

    Article Title: RIPK1-RIPK3-MLKL-dependent necrosis promotes the aging of mouse male reproductive system

    Journal: eLife

    doi: 10.7554/eLife.27692

    ( A, B ) Macroscopic features and weights of Ripk 3 +/+ (wild-type) and Ripk 3 -/- ( Ripk3 -knockout) male mice. Ripk 3 +/+ (4 Month, n = 16; 18 Month, n = 27) and Ripk 3 -/- (4 Month, n = 16; 18 Month, n = 27) male mice were photographed and weighed. Data represent the mean ±the standard error of the mean (s.e.m). **p<0.01, ***p<0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( C, D ) Macroscopic features and weights of seminal vesicles. Mice were sacrificed at 18 months of age, and the seminal vesicles from Ripk 3 +/+ (n = 33) and Ripk 3 -/- (n = 30) mice were photographed and weighed. Data represent the mean ±s.e.m. ***p < 0.001. p values were determined with unpaired Student’s t -tests. ( E ) Serum testosterone levels of mice assayed using ELISA. Mice were sacrificed, and the testosterone levels in serum from Ripk 3 +/+ (4 Month, n = 9; 18 Month, n = 9) and Ripk 3 -/- (4 Month, n = 9; 18 Month, n = 9) mice were measured using an ELISA kit for testosterone. Data represent the mean ±s.e.m. *p<0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( F, G ) H and E of testis sections from Ripk 3 +/+ and Ripk 3 -/- mice. Ripk 3 +/+ (4 Months, n = 10; 18 Months, n = 10) and Ripk 3 -/- (4 Months, n = 10; 18 Months, n = 10) mice were sacrificed and testes were harvested and stained with H and E in ( F ). The number of empty seminiferous tubules was counted based on H and E staining and quantification in ( G ), empty seminiferous tubules were counted in five fields per testis. Scale bar, 100 μm. Data represent the mean ± S.D. ***p < 0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( H ) Summary of the fertility rates of Ripk 3 +/+ and Ripk 3 -/- mice. One male mice of a given age was mated with a pairs of 10-week-old wild-type female mice for 3 months; females were replaced every 2 weeks. The number of male mice with reproductive capacity was counted (see Materials and methods). p values were determined using chi-square tests. ( I ) Reproductive longevity. When Ripk 3 +/+ (n = 12) and Ripk 3 -/- (n = 12) male mice were 2 months old, they were continuously mated with a pairs of 10-week-old female mice until pregnancies ceased; females were replaced every 2 months. The ages of the males at which their last litter was sired was recorded (calculated as the age at birth of the litter less 21 days, see Materials and methods). Data represent the mean ± S.D. **p < 0.01. p values were determined with unpaired Student’s t -tests. DOI: http://dx.doi.org/10.7554/eLife.27692.002 10.7554/eLife.27692.003 Figure 1—source data 1. Summary of the fertility rates and mortality rates of the offspring of 4- or 18-month-old Ripk 3 +/+ and Ripk 3 -/- male mice. DOI: http://dx.doi.org/10.7554/eLife.27692.003 10.7554/eLife.27692.004 Figure 1—source data 2. Summary of the fertility rates and mortality rates of the offspring of 13-month-old Ripk 3 +/+ and Ripk 3 -/- male mice. DOI: http://dx.doi.org/10.7554/eLife.27692.004
    Figure Legend Snippet: ( A, B ) Macroscopic features and weights of Ripk 3 +/+ (wild-type) and Ripk 3 -/- ( Ripk3 -knockout) male mice. Ripk 3 +/+ (4 Month, n = 16; 18 Month, n = 27) and Ripk 3 -/- (4 Month, n = 16; 18 Month, n = 27) male mice were photographed and weighed. Data represent the mean ±the standard error of the mean (s.e.m). **p<0.01, ***p<0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( C, D ) Macroscopic features and weights of seminal vesicles. Mice were sacrificed at 18 months of age, and the seminal vesicles from Ripk 3 +/+ (n = 33) and Ripk 3 -/- (n = 30) mice were photographed and weighed. Data represent the mean ±s.e.m. ***p < 0.001. p values were determined with unpaired Student’s t -tests. ( E ) Serum testosterone levels of mice assayed using ELISA. Mice were sacrificed, and the testosterone levels in serum from Ripk 3 +/+ (4 Month, n = 9; 18 Month, n = 9) and Ripk 3 -/- (4 Month, n = 9; 18 Month, n = 9) mice were measured using an ELISA kit for testosterone. Data represent the mean ±s.e.m. *p<0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( F, G ) H and E of testis sections from Ripk 3 +/+ and Ripk 3 -/- mice. Ripk 3 +/+ (4 Months, n = 10; 18 Months, n = 10) and Ripk 3 -/- (4 Months, n = 10; 18 Months, n = 10) mice were sacrificed and testes were harvested and stained with H and E in ( F ). The number of empty seminiferous tubules was counted based on H and E staining and quantification in ( G ), empty seminiferous tubules were counted in five fields per testis. Scale bar, 100 μm. Data represent the mean ± S.D. ***p < 0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( H ) Summary of the fertility rates of Ripk 3 +/+ and Ripk 3 -/- mice. One male mice of a given age was mated with a pairs of 10-week-old wild-type female mice for 3 months; females were replaced every 2 weeks. The number of male mice with reproductive capacity was counted (see Materials and methods). p values were determined using chi-square tests. ( I ) Reproductive longevity. When Ripk 3 +/+ (n = 12) and Ripk 3 -/- (n = 12) male mice were 2 months old, they were continuously mated with a pairs of 10-week-old female mice until pregnancies ceased; females were replaced every 2 months. The ages of the males at which their last litter was sired was recorded (calculated as the age at birth of the litter less 21 days, see Materials and methods). Data represent the mean ± S.D. **p < 0.01. p values were determined with unpaired Student’s t -tests. DOI: http://dx.doi.org/10.7554/eLife.27692.002 10.7554/eLife.27692.003 Figure 1—source data 1. Summary of the fertility rates and mortality rates of the offspring of 4- or 18-month-old Ripk 3 +/+ and Ripk 3 -/- male mice. DOI: http://dx.doi.org/10.7554/eLife.27692.003 10.7554/eLife.27692.004 Figure 1—source data 2. Summary of the fertility rates and mortality rates of the offspring of 13-month-old Ripk 3 +/+ and Ripk 3 -/- male mice. DOI: http://dx.doi.org/10.7554/eLife.27692.004

    Techniques Used: Knock-Out, Enzyme-linked Immunosorbent Assay, Staining

    ( A ) Mortality of offspring from Ripk 3 +/+ and Ripk 3 -/- male mice. One male mice of a given age was mated with a pairs of 10-week-old female wild-type mice for 3 months; females were replaced every 2 weeks. Litters were counted by date of birth of the pups; if a litter was born but did not survive, we counted the dead pups; if we were not able to count the pups, the number of pups was entered as ‘0’. Mortality of offspring from 4-month-old Ripk 3 +/+ (n = 5) and Ripk 3 -/- (n = 5) male mice; offspring from 18-month-old Ripk3 +/+ (n = 4) and Ripk 3 -/- (n = 15) male mice were calculated. Data represent the mean ± S.D. **p < 0.01, ***p < 0.001. P values were determined with unpaired Student’s t -tests. NS, not significant. ( B, C ) IHC of Ripk 3 +/+ and Ripk 3 -/- testes with 8-OHdG antibody. Mice were sacrificed, testes from Ripk 3 +/+ (4 Months, n = 6; 18 Months, n = 6) and Ripk 3 -/- (4 Months, n = 6; 18 Months, n = 6) mice were harvested and stained with 8-OHdG antibody in ( B ) (black arrows for sperm with 8-OHdG staining). 8-OHdG + sperm were counted in five fields per testis and quantification in ( C ). Scale bar, 100 μm. Data represent the mean ± S.D. ***p < 0.001. p values were determined with unpaired Student’s t-tests. DOI: http://dx.doi.org/10.7554/eLife.27692.011
    Figure Legend Snippet: ( A ) Mortality of offspring from Ripk 3 +/+ and Ripk 3 -/- male mice. One male mice of a given age was mated with a pairs of 10-week-old female wild-type mice for 3 months; females were replaced every 2 weeks. Litters were counted by date of birth of the pups; if a litter was born but did not survive, we counted the dead pups; if we were not able to count the pups, the number of pups was entered as ‘0’. Mortality of offspring from 4-month-old Ripk 3 +/+ (n = 5) and Ripk 3 -/- (n = 5) male mice; offspring from 18-month-old Ripk3 +/+ (n = 4) and Ripk 3 -/- (n = 15) male mice were calculated. Data represent the mean ± S.D. **p < 0.01, ***p < 0.001. P values were determined with unpaired Student’s t -tests. NS, not significant. ( B, C ) IHC of Ripk 3 +/+ and Ripk 3 -/- testes with 8-OHdG antibody. Mice were sacrificed, testes from Ripk 3 +/+ (4 Months, n = 6; 18 Months, n = 6) and Ripk 3 -/- (4 Months, n = 6; 18 Months, n = 6) mice were harvested and stained with 8-OHdG antibody in ( B ) (black arrows for sperm with 8-OHdG staining). 8-OHdG + sperm were counted in five fields per testis and quantification in ( C ). Scale bar, 100 μm. Data represent the mean ± S.D. ***p < 0.001. p values were determined with unpaired Student’s t-tests. DOI: http://dx.doi.org/10.7554/eLife.27692.011

    Techniques Used: Staining

    ( A ) Immunofluorescence of testes from Ripk 3 +/+ and Ripk 3 -/- mice (2 weeks) with RIPK3 antibody. Counterstaining with DAPI, blue. Scale bar, 100 μm. ( B ) No p-MLKL signaling in the testes from aged Mlkl -/- mice. WT (18 Months, n = 3) and Mlkl -/- (18 Months, n = 3) mice were sacrificed and testes were harvested and stained with p-MLKL antibody (black arrows indicate cells with p-MLKL staining). Scale bar, 100 μm. DOI: http://dx.doi.org/10.7554/eLife.27692.014
    Figure Legend Snippet: ( A ) Immunofluorescence of testes from Ripk 3 +/+ and Ripk 3 -/- mice (2 weeks) with RIPK3 antibody. Counterstaining with DAPI, blue. Scale bar, 100 μm. ( B ) No p-MLKL signaling in the testes from aged Mlkl -/- mice. WT (18 Months, n = 3) and Mlkl -/- (18 Months, n = 3) mice were sacrificed and testes were harvested and stained with p-MLKL antibody (black arrows indicate cells with p-MLKL staining). Scale bar, 100 μm. DOI: http://dx.doi.org/10.7554/eLife.27692.014

    Techniques Used: Immunofluorescence, Staining

    ( A ) RIPK3 expression in spermatogonia, Sertoli cells, and spermatocytes. Immunofluorescence in an 8-week-old testis with antibodies against RIPK3 (red), HSD3B1 (Leydig cells specific protein, green), GATA-1 (Sertoli cells specific protein, green), and UTF1 (spermatogonium specific protein, green). Scale bar, 100 μm. ( B ) RIPK3 expression in germ line cells and Sertoli cells. Primary testis cells were isolated from wild-type testes, Immunofluorescence of Leydig cells, Sertoli cells, spermatogonia, and primary spermatocytes with antibodies against RIPK3 (red), HSD3B1 (green), GATA-1 (green), UTF1 (green), and SMAD3 (primary spermatocytes specific protein, green). Counterstaining with DAPI, blue. Scale bar, 10 μm. ( C, D ) Immunohistochemistry (IHC) of testes from Ripk 3 +/+ and Ripk 3 -/- mice with phosphor-MLKL (p-MLKL) antibody. Ripk 3 +/+ (4 Months, n = 6; 18 Months, n = 6) and Ripk 3 -/- (4 Months, n = 6; 18 Months, n = 6) mice were sacrificed and testes were harvested and stained with p-MLKL antibody in ( C ) (black arrows indicate cells with p-MLKL staining). p-MLKL + cells were counted in five fields per testis and quantification in ( D ). Scale bar, 100 μm. Data represent the mean ± s.e.m. ***p<0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( E ) Western blot analysis of RIPK1, RIPK3, MLKL, and p-MLKL levels in the testis after perfusion, each group is representative of at least three mice. GAPDH was used as loading control. The asterisk (*) indicates non-specific bands. ( F ) Immunofluorescence in an 18-month-old testis with antibodies against p-MLKL (red, purple arrows indicate spermatogonium with p-MLKL staining), HSD3B1, GATA-1, and UTF1. Scale bar, 50 μm. DOI: http://dx.doi.org/10.7554/eLife.27692.013
    Figure Legend Snippet: ( A ) RIPK3 expression in spermatogonia, Sertoli cells, and spermatocytes. Immunofluorescence in an 8-week-old testis with antibodies against RIPK3 (red), HSD3B1 (Leydig cells specific protein, green), GATA-1 (Sertoli cells specific protein, green), and UTF1 (spermatogonium specific protein, green). Scale bar, 100 μm. ( B ) RIPK3 expression in germ line cells and Sertoli cells. Primary testis cells were isolated from wild-type testes, Immunofluorescence of Leydig cells, Sertoli cells, spermatogonia, and primary spermatocytes with antibodies against RIPK3 (red), HSD3B1 (green), GATA-1 (green), UTF1 (green), and SMAD3 (primary spermatocytes specific protein, green). Counterstaining with DAPI, blue. Scale bar, 10 μm. ( C, D ) Immunohistochemistry (IHC) of testes from Ripk 3 +/+ and Ripk 3 -/- mice with phosphor-MLKL (p-MLKL) antibody. Ripk 3 +/+ (4 Months, n = 6; 18 Months, n = 6) and Ripk 3 -/- (4 Months, n = 6; 18 Months, n = 6) mice were sacrificed and testes were harvested and stained with p-MLKL antibody in ( C ) (black arrows indicate cells with p-MLKL staining). p-MLKL + cells were counted in five fields per testis and quantification in ( D ). Scale bar, 100 μm. Data represent the mean ± s.e.m. ***p<0.001. p values were determined with unpaired Student’s t -tests. NS, not significant. ( E ) Western blot analysis of RIPK1, RIPK3, MLKL, and p-MLKL levels in the testis after perfusion, each group is representative of at least three mice. GAPDH was used as loading control. The asterisk (*) indicates non-specific bands. ( F ) Immunofluorescence in an 18-month-old testis with antibodies against p-MLKL (red, purple arrows indicate spermatogonium with p-MLKL staining), HSD3B1, GATA-1, and UTF1. Scale bar, 50 μm. DOI: http://dx.doi.org/10.7554/eLife.27692.013

    Techniques Used: Expressing, Immunofluorescence, Isolation, Immunohistochemistry, Staining, Western Blot

    ( A, B ) IHC of testis from Ripk +/+ and Ripk 3 -/- mice with Cleaved-Caspase-3 antibody. Mice were sacrificed, testes from Ripk 3 +/+ (4 Month, n = 6; 18 Month, n = 6) and Ripk 3 -/- (4 Month, n = 6; 18 Month, n = 6) mice were harvested and stained with Cleaved-Caspase-3 antibody in ( A ) (black arrows for Leydig cells with Cleaved-Caspase-3 staining). Cleaved-Caspase-3 + cells were counted in six fields per testis and quantification in ( B ). Scale bar, 100 μm. Data represent the mean ± s.e.m. *p < 0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. ( C, D ) IHC of testis from Ripk +/+ and Ripk -/- mice with Cleaved-Caspase-8 antibody. Mice were sacrificed, testes from Ripk +/+ (4 Month, n = 6; 18 Month, n = 6) and Ripk 3 -/- (4 Month, n = 6; 18 Month, n = 6) mice were harvested and stained with Cleaved-caspase-8 antibody in ( C ) (black arrows for Leydig cells with Cleaved-caspase-8 staining). Cleaved-caspase-8 + cells were counted in six fields per testis and quantification in ( D ). Scale bar, 100 μm. Data represent the mean ± s.e.m. *p<0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. ( E, F ) Caspase8 levels decrease during aging in empty seminiferous tubules. Immunofluorescence of testes from 4-month-old and 18-month-old wild-type mice with caspase8 and RIPK3 antibody in ( E ). The caspase8 levels were quantified in ( F ). Counterstaining with DAPI, blue. Scale bar, 100 μm. DOI: http://dx.doi.org/10.7554/eLife.27692.015
    Figure Legend Snippet: ( A, B ) IHC of testis from Ripk +/+ and Ripk 3 -/- mice with Cleaved-Caspase-3 antibody. Mice were sacrificed, testes from Ripk 3 +/+ (4 Month, n = 6; 18 Month, n = 6) and Ripk 3 -/- (4 Month, n = 6; 18 Month, n = 6) mice were harvested and stained with Cleaved-Caspase-3 antibody in ( A ) (black arrows for Leydig cells with Cleaved-Caspase-3 staining). Cleaved-Caspase-3 + cells were counted in six fields per testis and quantification in ( B ). Scale bar, 100 μm. Data represent the mean ± s.e.m. *p < 0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. ( C, D ) IHC of testis from Ripk +/+ and Ripk -/- mice with Cleaved-Caspase-8 antibody. Mice were sacrificed, testes from Ripk +/+ (4 Month, n = 6; 18 Month, n = 6) and Ripk 3 -/- (4 Month, n = 6; 18 Month, n = 6) mice were harvested and stained with Cleaved-caspase-8 antibody in ( C ) (black arrows for Leydig cells with Cleaved-caspase-8 staining). Cleaved-caspase-8 + cells were counted in six fields per testis and quantification in ( D ). Scale bar, 100 μm. Data represent the mean ± s.e.m. *p<0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. ( E, F ) Caspase8 levels decrease during aging in empty seminiferous tubules. Immunofluorescence of testes from 4-month-old and 18-month-old wild-type mice with caspase8 and RIPK3 antibody in ( E ). The caspase8 levels were quantified in ( F ). Counterstaining with DAPI, blue. Scale bar, 100 μm. DOI: http://dx.doi.org/10.7554/eLife.27692.015

    Techniques Used: Staining, Immunofluorescence

    ( A, B ) IHC of testis from Ripk 3 +/+ and Ripk 3 -/- mice with Cleaved-Caspase-3 antibody. Mice were sacrificed, testes from Ripk 3 +/+ (4 Month, n = 6; 36 Months, n = 6) and Ripk 3 -/- (4 Month, n = 6; 36 Months, n = 6) mice were harvested and stained with Cleaved-Caspase-3 antibody in ( A ) (black arrows for Leydig cells with Cleaved-Caspase-3 staining). Cleaved-Caspase-3 + cells were counted in six fields per testis and quantification in ( B ). Scale bar, 100 μm. Data represent the mean ± s.e.m. *p<0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. ( C ) Western blot analysis of RIPK3 and Cleaved-caspase-3 levels in the testis after perfusion; each group is representative of at least three mice. GAPDH was used as a loading control. DOI: http://dx.doi.org/10.7554/eLife.27692.016
    Figure Legend Snippet: ( A, B ) IHC of testis from Ripk 3 +/+ and Ripk 3 -/- mice with Cleaved-Caspase-3 antibody. Mice were sacrificed, testes from Ripk 3 +/+ (4 Month, n = 6; 36 Months, n = 6) and Ripk 3 -/- (4 Month, n = 6; 36 Months, n = 6) mice were harvested and stained with Cleaved-Caspase-3 antibody in ( A ) (black arrows for Leydig cells with Cleaved-Caspase-3 staining). Cleaved-Caspase-3 + cells were counted in six fields per testis and quantification in ( B ). Scale bar, 100 μm. Data represent the mean ± s.e.m. *p<0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. ( C ) Western blot analysis of RIPK3 and Cleaved-caspase-3 levels in the testis after perfusion; each group is representative of at least three mice. GAPDH was used as a loading control. DOI: http://dx.doi.org/10.7554/eLife.27692.016

    Techniques Used: Staining, Western Blot

    ( A ) Western blot analysis of RIPK1, RIPK3, MLKL, and p-MLKL levels in testes after injection with TSZ. The 2-month-old wild-type male mice continuously feed with RIPA-56 (0 mg/kg, n = 6; 150 mg/kg, n = 3; 300 mg/kg, n = 3) for one week. Testes were injected with TSZ (see Materials and methods); 72 hr after the injection, mice were sacrificed and the testes were harvested. The proteins were extracted from testes and were analyzed with western blotting. GAPDH was used as a loading control. ( B–H ) 13-month-old wild-type male mice were feed with AIN93G (RIPA-56:0 mg/kg) or AIN93G-RIPA-56 (RIPA-56:300 mg/kg) for 2 months in SPF facility. Mice were weighed before and after feed with RIPA-56 in ( B ). Data represent the mean ± s.e.m. *p<0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. Mice were sacrificed and the seminal vesicles were photographed and weighed. Macroscopic features and weights of seminal vesicles form mice in ( C, D ). Data represent the mean ± s.e.m. ***p<0.001. p values were determined with unpaired Student’s t -tests. Testosterone levels in serum from mice were measured using a testosterone ELISA kit in ( E ). Data represent the mean ± s.e.m. **p<0.01. p values were determined with unpaired Student’s t -tests. The testes were harvested and stained with H and E in ( F ). The number of empty seminiferous tubules was counted based on H and E staining and quantification in ( G ). Empty seminiferous tubules were counted in five fields per testis. Scale bar, 100 μm. Data represent the mean ± S.D. **p<0.01. p values were determined with unpaired Student’s t -tests. The fertility rate of each RIPA-56-treated (0 mg/kg, n = 23; 300 mg/kg, n = 25) male mice was assessed by mating it with four 10-week-old wild-type female mice successively (see Materials and methods). The number of mice with reproductive capacity was counted in ( H ). p values were determined using chi-square tests. ( I–K ) 13-month-old wild-type male mice were feed with AIN93G (RIPA-56:0 mg/kg) or AIN93G-RIPA-56 (RIPA-56:300 mg/kg) for 5 months in SPF facility. The fertility rate of each RIPA-56-treated (0 mg/kg, n = 15; 300 mg/kg, n = 15) male mice was assessed by mating it with four 10-week-old wild-type female mice successively (see Materials and methods). The number of mice with reproductive capacity was counted in ( I ). p values were determined using chi-square tests. After fertility test, mice were sacrificed and testes were harvested and stained with p-MLKL antibody in ( J ) (black arrows indicate cells with p-MLKL staining). p-MLKL + cells were counted in five fields per testis and quantification in ( K ). Scale bar, 100 μm. Data represent the mean ± s.e.m. ***p<0.001. P values were determined with unpaired Student’s t -tests. DOI: http://dx.doi.org/10.7554/eLife.27692.022
    Figure Legend Snippet: ( A ) Western blot analysis of RIPK1, RIPK3, MLKL, and p-MLKL levels in testes after injection with TSZ. The 2-month-old wild-type male mice continuously feed with RIPA-56 (0 mg/kg, n = 6; 150 mg/kg, n = 3; 300 mg/kg, n = 3) for one week. Testes were injected with TSZ (see Materials and methods); 72 hr after the injection, mice were sacrificed and the testes were harvested. The proteins were extracted from testes and were analyzed with western blotting. GAPDH was used as a loading control. ( B–H ) 13-month-old wild-type male mice were feed with AIN93G (RIPA-56:0 mg/kg) or AIN93G-RIPA-56 (RIPA-56:300 mg/kg) for 2 months in SPF facility. Mice were weighed before and after feed with RIPA-56 in ( B ). Data represent the mean ± s.e.m. *p<0.05, ***p<0.001. p values were determined with unpaired Student’s t -tests. Mice were sacrificed and the seminal vesicles were photographed and weighed. Macroscopic features and weights of seminal vesicles form mice in ( C, D ). Data represent the mean ± s.e.m. ***p<0.001. p values were determined with unpaired Student’s t -tests. Testosterone levels in serum from mice were measured using a testosterone ELISA kit in ( E ). Data represent the mean ± s.e.m. **p<0.01. p values were determined with unpaired Student’s t -tests. The testes were harvested and stained with H and E in ( F ). The number of empty seminiferous tubules was counted based on H and E staining and quantification in ( G ). Empty seminiferous tubules were counted in five fields per testis. Scale bar, 100 μm. Data represent the mean ± S.D. **p<0.01. p values were determined with unpaired Student’s t -tests. The fertility rate of each RIPA-56-treated (0 mg/kg, n = 23; 300 mg/kg, n = 25) male mice was assessed by mating it with four 10-week-old wild-type female mice successively (see Materials and methods). The number of mice with reproductive capacity was counted in ( H ). p values were determined using chi-square tests. ( I–K ) 13-month-old wild-type male mice were feed with AIN93G (RIPA-56:0 mg/kg) or AIN93G-RIPA-56 (RIPA-56:300 mg/kg) for 5 months in SPF facility. The fertility rate of each RIPA-56-treated (0 mg/kg, n = 15; 300 mg/kg, n = 15) male mice was assessed by mating it with four 10-week-old wild-type female mice successively (see Materials and methods). The number of mice with reproductive capacity was counted in ( I ). p values were determined using chi-square tests. After fertility test, mice were sacrificed and testes were harvested and stained with p-MLKL antibody in ( J ) (black arrows indicate cells with p-MLKL staining). p-MLKL + cells were counted in five fields per testis and quantification in ( K ). Scale bar, 100 μm. Data represent the mean ± s.e.m. ***p<0.001. P values were determined with unpaired Student’s t -tests. DOI: http://dx.doi.org/10.7554/eLife.27692.022

    Techniques Used: Western Blot, Injection, Enzyme-linked Immunosorbent Assay, Staining

    Western blot analysis of TNF-α, RIPK1, RIPK3, MLKL, Cleaved-caspase3 and p-MLKL levels in the testis after perfusion, each group is representative of at least three mice. GAPDH was used as loading control. DOI: http://dx.doi.org/10.7554/eLife.27692.023
    Figure Legend Snippet: Western blot analysis of TNF-α, RIPK1, RIPK3, MLKL, Cleaved-caspase3 and p-MLKL levels in the testis after perfusion, each group is representative of at least three mice. GAPDH was used as loading control. DOI: http://dx.doi.org/10.7554/eLife.27692.023

    Techniques Used: Western Blot

    antibody against ripk3  (ProSci Incorporated)


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    ProSci Incorporated antibody against ripk3
    Antibody Against 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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    ProSci Incorporated rip3
    (A) Nec-1 inhibits TNF-induced necrosis in L929 cells. L929 cells were treated with 10 µM zVAD-fmk, 20 µM Nec-1 and 10 ng/ml mouse TNF (mTNF) as indicated. Cell death was determined by MTS assay as described in materials and methods. (B) L929 cells were treated with the indicated doses of Nec-1, followed by stimulation with 10 ng/ml mTNF. Cell Death was analyzed as in (A). (C) Nec-1 did not alter RIP1 or <t>RIP3</t> protein expression in L929 cells.
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    The regulation of <t>RIP3</t> in TNF- α -induced toxicity of hippocampal neurons in vivo . (a) Nissl staining of hippocampal neurons 72 h after treatment. Wild-type (WT) and RIP3 knockout (KO) mice received intracerebroventricular injection of PBS or the indicated dose of TNF- α . The neurons of brain sections from WT and KO mice were analyzed by Nissl staining ( n = 7) and morphology of hippocampal CA3 region was shown. Arrows indicate the loss of hippocampal neurons. (b) and (c) Expressions of RIP1, RIP3, and caspase-3 in the hippocampus after TNF - α treatment. Proteins extracted from hippocampus in the wild-type mice treated with PBS or TNF- α were analyzed by western blot using indicated antibodies. PC: MEF cells were treated with staurosporine at 150 nM for 15 hours. The results shown here are representative of five mice.
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    ( A – C ) Immunoblot analysis of ( A ) pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P53) and activated (P34) gasdermin E (GSDME); ( B ) pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17) and pro- (P35) and cleaved caspase-7 (CASP7; P20); and ( C ) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated <t>RIPK3</t> (pRIPK3) and total RIPK3 (tRIPK3) in the lung samples from mock- or IFN-β–treated wild type (WT) mice with or without mouse hepatitis virus (MHV) infection 3 days post-infection. ( D – I ) Immunoblot analysis of ( D , G ) pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) GSDMD, pro- (P53) and activated (P34) GSDME; ( E , H ) pro- (P55) and cleaved CASP8 (P18), pro- (P35) and cleaved CASP3 (P19 and P17) and pro- (P35) and cleaved CASP7 (P20); ( F) pMLKL, tMLKL, pRIPK3 and tRIPK3; and ( I ) pRIPK3 and tRIPK3 in mock- or IFN-β–treated bone marrow-derived macrophages (BMDMs) or THP-1 cells during MHV or SARS-CoV-2 infection, respectively. Actin was used as the internal control. Molecular weight marker sizes in kDa are indicated in small font on the left of each blot. Asterisk denotes non-specific bands ( A, G ). Data are representative of at least three independent experiments.
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    ( A – C ) Immunoblot analysis of ( A ) pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P53) and activated (P34) gasdermin E (GSDME); ( B ) pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17) and pro- (P35) and cleaved caspase-7 (CASP7; P20); and ( C ) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated <t>RIPK3</t> (pRIPK3) and total RIPK3 (tRIPK3) in the lung samples from mock- or IFN-β–treated wild type (WT) mice with or without mouse hepatitis virus (MHV) infection 3 days post-infection. ( D – I ) Immunoblot analysis of ( D , G ) pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) GSDMD, pro- (P53) and activated (P34) GSDME; ( E , H ) pro- (P55) and cleaved CASP8 (P18), pro- (P35) and cleaved CASP3 (P19 and P17) and pro- (P35) and cleaved CASP7 (P20); ( F) pMLKL, tMLKL, pRIPK3 and tRIPK3; and ( I ) pRIPK3 and tRIPK3 in mock- or IFN-β–treated bone marrow-derived macrophages (BMDMs) or THP-1 cells during MHV or SARS-CoV-2 infection, respectively. Actin was used as the internal control. Molecular weight marker sizes in kDa are indicated in small font on the left of each blot. Asterisk denotes non-specific bands ( A, G ). Data are representative of at least three independent experiments.
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    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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    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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    Image Search Results


    (A) Nec-1 inhibits TNF-induced necrosis in L929 cells. L929 cells were treated with 10 µM zVAD-fmk, 20 µM Nec-1 and 10 ng/ml mouse TNF (mTNF) as indicated. Cell death was determined by MTS assay as described in materials and methods. (B) L929 cells were treated with the indicated doses of Nec-1, followed by stimulation with 10 ng/ml mTNF. Cell Death was analyzed as in (A). (C) Nec-1 did not alter RIP1 or RIP3 protein expression in L929 cells.

    Journal: PLoS ONE

    Article Title: RIP1-Dependent and Independent Effects of Necrostatin-1 in Necrosis and T Cell Activation

    doi: 10.1371/journal.pone.0023209

    Figure Lengend Snippet: (A) Nec-1 inhibits TNF-induced necrosis in L929 cells. L929 cells were treated with 10 µM zVAD-fmk, 20 µM Nec-1 and 10 ng/ml mouse TNF (mTNF) as indicated. Cell death was determined by MTS assay as described in materials and methods. (B) L929 cells were treated with the indicated doses of Nec-1, followed by stimulation with 10 ng/ml mTNF. Cell Death was analyzed as in (A). (C) Nec-1 did not alter RIP1 or RIP3 protein expression in L929 cells.

    Article Snippet: After transfer to nitrocellulose membranes, Western blots were performed with antibodies to RIP1, §-actin, PARP-1 (BD Pharmingen, San Diego, CA, USA), RIP3 (ProSci, Poway, CA, USA), Erk, Jnk, PKA substrate (Cell Signaling, Danvers, MA, USA), PKA, LAT (Santa Cruz, Santa Cruz, CA, USA), and phospho-tyrosine (Millipore, Bellerica, MA, USA).

    Techniques: MTS Assay, Expressing

    (A–C) RIP1 is required for zVAD-fmk, but not TNF-induced necrosis in L929 cells. (A) L929 cells were transfected with the indicated siRNA oligonucleotides with HiPerfect (Qiagen). TR4 is the control siRNA against TRAIL-R4 . Cells were treated with (A) 50 µM zVAD-fmk or (B) 10 ng/ml mTNF and cell death was measured by MTS assay. (C) Western blot shows RIP1 and RIP3 expression in cells transfected with the indicated siRNAs. (D–E) RIP1 is required for TNF-induced necrosis in 3T3 fibroblasts. NIH 3T3 fibroblasts were transfected with the indicated siRNA. (D) Expression of RIP1, RIP3 and ß-actin in cells transfected with the indicated siRNA was determined by Western blot. (E) Necrosis was induced with 10 µM zVAD-fmk, 0.5 µg/ml cycloheximide and 10 ng/ml mTNF for 20 hours. Cell death was assessed as in (A).

    Journal: PLoS ONE

    Article Title: RIP1-Dependent and Independent Effects of Necrostatin-1 in Necrosis and T Cell Activation

    doi: 10.1371/journal.pone.0023209

    Figure Lengend Snippet: (A–C) RIP1 is required for zVAD-fmk, but not TNF-induced necrosis in L929 cells. (A) L929 cells were transfected with the indicated siRNA oligonucleotides with HiPerfect (Qiagen). TR4 is the control siRNA against TRAIL-R4 . Cells were treated with (A) 50 µM zVAD-fmk or (B) 10 ng/ml mTNF and cell death was measured by MTS assay. (C) Western blot shows RIP1 and RIP3 expression in cells transfected with the indicated siRNAs. (D–E) RIP1 is required for TNF-induced necrosis in 3T3 fibroblasts. NIH 3T3 fibroblasts were transfected with the indicated siRNA. (D) Expression of RIP1, RIP3 and ß-actin in cells transfected with the indicated siRNA was determined by Western blot. (E) Necrosis was induced with 10 µM zVAD-fmk, 0.5 µg/ml cycloheximide and 10 ng/ml mTNF for 20 hours. Cell death was assessed as in (A).

    Article Snippet: After transfer to nitrocellulose membranes, Western blots were performed with antibodies to RIP1, §-actin, PARP-1 (BD Pharmingen, San Diego, CA, USA), RIP3 (ProSci, Poway, CA, USA), Erk, Jnk, PKA substrate (Cell Signaling, Danvers, MA, USA), PKA, LAT (Santa Cruz, Santa Cruz, CA, USA), and phospho-tyrosine (Millipore, Bellerica, MA, USA).

    Techniques: Transfection, MTS Assay, Western Blot, Expressing

    The regulation of RIP3 in TNF- α -induced toxicity of hippocampal neurons in vivo . (a) Nissl staining of hippocampal neurons 72 h after treatment. Wild-type (WT) and RIP3 knockout (KO) mice received intracerebroventricular injection of PBS or the indicated dose of TNF- α . The neurons of brain sections from WT and KO mice were analyzed by Nissl staining ( n = 7) and morphology of hippocampal CA3 region was shown. Arrows indicate the loss of hippocampal neurons. (b) and (c) Expressions of RIP1, RIP3, and caspase-3 in the hippocampus after TNF - α treatment. Proteins extracted from hippocampus in the wild-type mice treated with PBS or TNF- α were analyzed by western blot using indicated antibodies. PC: MEF cells were treated with staurosporine at 150 nM for 15 hours. The results shown here are representative of five mice.

    Journal: BioMed Research International

    Article Title: Necroptosis Mediates TNF-Induced Toxicity of Hippocampal Neurons

    doi: 10.1155/2014/290182

    Figure Lengend Snippet: The regulation of RIP3 in TNF- α -induced toxicity of hippocampal neurons in vivo . (a) Nissl staining of hippocampal neurons 72 h after treatment. Wild-type (WT) and RIP3 knockout (KO) mice received intracerebroventricular injection of PBS or the indicated dose of TNF- α . The neurons of brain sections from WT and KO mice were analyzed by Nissl staining ( n = 7) and morphology of hippocampal CA3 region was shown. Arrows indicate the loss of hippocampal neurons. (b) and (c) Expressions of RIP1, RIP3, and caspase-3 in the hippocampus after TNF - α treatment. Proteins extracted from hippocampus in the wild-type mice treated with PBS or TNF- α were analyzed by western blot using indicated antibodies. PC: MEF cells were treated with staurosporine at 150 nM for 15 hours. The results shown here are representative of five mice.

    Article Snippet: The following antibodies were used for western blotting: mouse RIP3 (Prosci, 2283), RIP1 (BD Biosciences, 610459), mouse CYLD (Cell Signaling, 437700), caspase-3 (Cell Signaling, 9662), and β -actin (Sigma).

    Techniques: In Vivo, Staining, Knock-Out, Injection, Western Blot

    TNF- α -induced necrosis of HT-22 cells depends on RIP3 and its kinase activity. (a) HT-22 cells were transfected with the negative control (NC) or RIP3 siRNAs. After 60 h, cells were treated with control or TNF- α /z-VAD for another 20 h and then cell viability was determined by measuring ATP levels. Data were represented as mean ± standard deviation of duplicates. * P < 0.01, ** P < 0.001 versus NC-T + Z. (b) The knockdown efficiency of RIP3 RNAi. Cell lysates were collected 60 h after transfection and subjected to western blot analysis of RIP3 and β -actin levels. (c) HT-22 cells stably expressing a siRNA-resistant WT-RIP3 or RIP3-K51A or RIP3-RHIM-Mut were transfected with the control or RIP3 siRNAs. After 60 h, cells were treated with control or TNF- α /z-VAD for 20 h and then cell viability was determined by measuring ATP levels. Data were represented as mean ± standard deviation of duplicates. * P < 0.01, ** P < 0.001 versus NC-T + Z. WT-RIP3: HT-22 cells stably expressing a siRNA-resistant wild-type form of RIP3; RIP3-K51A: HT-22 cells stably expressing a siRNA-resistant RIP3 kinase dead mutant. RIP3-RHIM Mut: HT-22 cells stably expressing a siRNA-resistant RHIM domain mutant form of RIP3. (d) The knockdown efficiency of RIP3 RNAi. Cell lysates were collected 60 h after transfection and subjected to western blot analysis of RIP3 and β -actin levels. All experiments were repeated three times with similar results.

    Journal: BioMed Research International

    Article Title: Necroptosis Mediates TNF-Induced Toxicity of Hippocampal Neurons

    doi: 10.1155/2014/290182

    Figure Lengend Snippet: TNF- α -induced necrosis of HT-22 cells depends on RIP3 and its kinase activity. (a) HT-22 cells were transfected with the negative control (NC) or RIP3 siRNAs. After 60 h, cells were treated with control or TNF- α /z-VAD for another 20 h and then cell viability was determined by measuring ATP levels. Data were represented as mean ± standard deviation of duplicates. * P < 0.01, ** P < 0.001 versus NC-T + Z. (b) The knockdown efficiency of RIP3 RNAi. Cell lysates were collected 60 h after transfection and subjected to western blot analysis of RIP3 and β -actin levels. (c) HT-22 cells stably expressing a siRNA-resistant WT-RIP3 or RIP3-K51A or RIP3-RHIM-Mut were transfected with the control or RIP3 siRNAs. After 60 h, cells were treated with control or TNF- α /z-VAD for 20 h and then cell viability was determined by measuring ATP levels. Data were represented as mean ± standard deviation of duplicates. * P < 0.01, ** P < 0.001 versus NC-T + Z. WT-RIP3: HT-22 cells stably expressing a siRNA-resistant wild-type form of RIP3; RIP3-K51A: HT-22 cells stably expressing a siRNA-resistant RIP3 kinase dead mutant. RIP3-RHIM Mut: HT-22 cells stably expressing a siRNA-resistant RHIM domain mutant form of RIP3. (d) The knockdown efficiency of RIP3 RNAi. Cell lysates were collected 60 h after transfection and subjected to western blot analysis of RIP3 and β -actin levels. All experiments were repeated three times with similar results.

    Article Snippet: The following antibodies were used for western blotting: mouse RIP3 (Prosci, 2283), RIP1 (BD Biosciences, 610459), mouse CYLD (Cell Signaling, 437700), caspase-3 (Cell Signaling, 9662), and β -actin (Sigma).

    Techniques: Activity Assay, Transfection, Negative Control, Standard Deviation, Western Blot, Stable Transfection, Expressing, Mutagenesis

    ( A – C ) Immunoblot analysis of ( A ) pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P53) and activated (P34) gasdermin E (GSDME); ( B ) pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17) and pro- (P35) and cleaved caspase-7 (CASP7; P20); and ( C ) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated RIPK3 (pRIPK3) and total RIPK3 (tRIPK3) in the lung samples from mock- or IFN-β–treated wild type (WT) mice with or without mouse hepatitis virus (MHV) infection 3 days post-infection. ( D – I ) Immunoblot analysis of ( D , G ) pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) GSDMD, pro- (P53) and activated (P34) GSDME; ( E , H ) pro- (P55) and cleaved CASP8 (P18), pro- (P35) and cleaved CASP3 (P19 and P17) and pro- (P35) and cleaved CASP7 (P20); ( F) pMLKL, tMLKL, pRIPK3 and tRIPK3; and ( I ) pRIPK3 and tRIPK3 in mock- or IFN-β–treated bone marrow-derived macrophages (BMDMs) or THP-1 cells during MHV or SARS-CoV-2 infection, respectively. Actin was used as the internal control. Molecular weight marker sizes in kDa are indicated in small font on the left of each blot. Asterisk denotes non-specific bands ( A, G ). Data are representative of at least three independent experiments.

    Journal: Science Immunology

    Article Title: ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection

    doi: 10.1126/sciimmunol.abo6294

    Figure Lengend Snippet: ( A – C ) Immunoblot analysis of ( A ) pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P53) and activated (P34) gasdermin E (GSDME); ( B ) pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17) and pro- (P35) and cleaved caspase-7 (CASP7; P20); and ( C ) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated RIPK3 (pRIPK3) and total RIPK3 (tRIPK3) in the lung samples from mock- or IFN-β–treated wild type (WT) mice with or without mouse hepatitis virus (MHV) infection 3 days post-infection. ( D – I ) Immunoblot analysis of ( D , G ) pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) GSDMD, pro- (P53) and activated (P34) GSDME; ( E , H ) pro- (P55) and cleaved CASP8 (P18), pro- (P35) and cleaved CASP3 (P19 and P17) and pro- (P35) and cleaved CASP7 (P20); ( F) pMLKL, tMLKL, pRIPK3 and tRIPK3; and ( I ) pRIPK3 and tRIPK3 in mock- or IFN-β–treated bone marrow-derived macrophages (BMDMs) or THP-1 cells during MHV or SARS-CoV-2 infection, respectively. Actin was used as the internal control. Molecular weight marker sizes in kDa are indicated in small font on the left of each blot. Asterisk denotes non-specific bands ( A, G ). Data are representative of at least three independent experiments.

    Article Snippet: Following electrophoretic transfer of proteins onto PVDF membranes (Millipore, IPVH00010), non-specific binding was blocked by incubation with 5% skim milk; then membranes were incubated with the following primary antibodies: anti-caspase-1 (AdipoGen, AG-20B-0042, 1:1000), anti-caspase-3 (CST, #9662, 1:1000), anti-cleaved caspase-3 (CST, #9661, 1:1000), anti-caspase-7 (CST, #9492, 1:1000), anti-cleaved caspase-7 (CST, #9491, 1:1000), anti-caspase-8 (CST, #4927, 1:1000), anti-cleaved caspase-8 (CST, #8592, 1:1000), anti-pRIPK3 (CST, #91702S, 1:1000), anti-RIPK3 (ProSci, #2283, 1:1000), anti-pMLKL (CST, #37333, 1:1000), anti-MLKL (Abgent, AP14272b, 1:1000), anti-GSDMD (Abcam, ab209845, 1:1000), anti-GSDME (Abcam, ab215191, 1:1000), anti-pSTAT1 (CST, #7649, 1:1000), anti-STAT1 (CST, #14994, 1:1000), anti-ZBP1 (AdipoGen, AG-20B-0010, 1:1000), anti-β-actin (Proteintech, 66009-1-IG, 1:5000), human anti-caspase-1 (R&D systems; Cat# MAB6215, 1:1000), human anti-caspase-8 (Enzo, ALX-804-242, 1:1000), human anti-GSDMD (Abcam, ab210070, 1:1000), human anti-tMLKL (Abcam, ab184718, 1:1000), human anti-β-actin (CST, 4970, 1:1000), human anti-ZBP1 (R&D systems; Cat# AF6309, 1:1000) and anti-ISG15 (Santa Cruz, sc-166755).

    Techniques: Western Blot, Infection, Derivative Assay, Molecular Weight, Marker

    ( A – C ) Immunoblot analysis of ( A ) pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P53) and activated (P34) gasdermin E (GSDME); ( B ) pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17) and pro- (P35) and cleaved caspase-7 (CASP7; P20); and ( C ) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated RIPK3 (pRIPK3) and total RIPK3 (tRIPK3) in the lung samples from PBS-treated mice or mouse hepatitis virus (MHV)-infected wild type (WT) and Zbp1 –/– mice treated with IFN-β harvested 3 days after infection. ( D – F ) Immunoblot analysis of ( D ) pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) GSDMD, pro- (P53) and activated (P34) GSDME; ( E ) pro- (P55) and cleaved (P18) CASP8, pro- (P35) and cleaved (P19 and P17) CASP3 and pro- (P35) and cleaved (P20) CASP7; and ( F ) pMLKL, tMLKL and ZBP1 in PBS- or IFN-β–treated WT, Zbp1 –/– and Zbp1 ∆Za2 bone marrow-derived macrophages (BMDMs) during MHV infection. Actin was used as the internal control. Data are representative of at least three independent experiments.

    Journal: Science Immunology

    Article Title: ZBP1-dependent inflammatory cell death, PANoptosis, and cytokine storm disrupt IFN therapeutic efficacy during coronavirus infection

    doi: 10.1126/sciimmunol.abo6294

    Figure Lengend Snippet: ( A – C ) Immunoblot analysis of ( A ) pro- (P53) and activated (P30) gasdermin D (GSDMD), pro- (P53) and activated (P34) gasdermin E (GSDME); ( B ) pro- (P55) and cleaved caspase-8 (CASP8; P18), pro- (P35) and cleaved caspase-3 (CASP3; P19 and P17) and pro- (P35) and cleaved caspase-7 (CASP7; P20); and ( C ) phosphorylated MLKL (pMLKL), total MLKL (tMLKL), phosphorylated RIPK3 (pRIPK3) and total RIPK3 (tRIPK3) in the lung samples from PBS-treated mice or mouse hepatitis virus (MHV)-infected wild type (WT) and Zbp1 –/– mice treated with IFN-β harvested 3 days after infection. ( D – F ) Immunoblot analysis of ( D ) pro- (P45) and activated (P20) caspase-1 (CASP1), pro- (P53) and activated (P30) GSDMD, pro- (P53) and activated (P34) GSDME; ( E ) pro- (P55) and cleaved (P18) CASP8, pro- (P35) and cleaved (P19 and P17) CASP3 and pro- (P35) and cleaved (P20) CASP7; and ( F ) pMLKL, tMLKL and ZBP1 in PBS- or IFN-β–treated WT, Zbp1 –/– and Zbp1 ∆Za2 bone marrow-derived macrophages (BMDMs) during MHV infection. Actin was used as the internal control. Data are representative of at least three independent experiments.

    Article Snippet: Following electrophoretic transfer of proteins onto PVDF membranes (Millipore, IPVH00010), non-specific binding was blocked by incubation with 5% skim milk; then membranes were incubated with the following primary antibodies: anti-caspase-1 (AdipoGen, AG-20B-0042, 1:1000), anti-caspase-3 (CST, #9662, 1:1000), anti-cleaved caspase-3 (CST, #9661, 1:1000), anti-caspase-7 (CST, #9492, 1:1000), anti-cleaved caspase-7 (CST, #9491, 1:1000), anti-caspase-8 (CST, #4927, 1:1000), anti-cleaved caspase-8 (CST, #8592, 1:1000), anti-pRIPK3 (CST, #91702S, 1:1000), anti-RIPK3 (ProSci, #2283, 1:1000), anti-pMLKL (CST, #37333, 1:1000), anti-MLKL (Abgent, AP14272b, 1:1000), anti-GSDMD (Abcam, ab209845, 1:1000), anti-GSDME (Abcam, ab215191, 1:1000), anti-pSTAT1 (CST, #7649, 1:1000), anti-STAT1 (CST, #14994, 1:1000), anti-ZBP1 (AdipoGen, AG-20B-0010, 1:1000), anti-β-actin (Proteintech, 66009-1-IG, 1:5000), human anti-caspase-1 (R&D systems; Cat# MAB6215, 1:1000), human anti-caspase-8 (Enzo, ALX-804-242, 1:1000), human anti-GSDMD (Abcam, ab210070, 1:1000), human anti-tMLKL (Abcam, ab184718, 1:1000), human anti-β-actin (CST, 4970, 1:1000), human anti-ZBP1 (R&D systems; Cat# AF6309, 1:1000) and anti-ISG15 (Santa Cruz, sc-166755).

    Techniques: Western Blot, Infection, Derivative Assay

    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