z vad fmk s7023  (Selleck Chemicals)


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    Selleck Chemicals z vad fmk s7023
    Z Vad Fmk S7023, supplied by Selleck Chemicals, 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/z vad fmk s7023/product/Selleck Chemicals
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    z vad fmk s7023 - by Bioz Stars, 2024-04
    86/100 stars

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    z vad fmk  (Selleck Chemicals)


    Bioz Manufacturer Symbol Selleck Chemicals manufactures this product  
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    Selleck Chemicals z vad fmk
    Compound heterozygous gain-of-function mutations in RIPK1 causes systemic autoinflammatory diseases. a , Pedigrees of a family with two patients carrying mutations at RIPK1 K377 and R390. Patient 1 (P1) and patient 2 (P2) were represented with filled symbols. b, Timeline of recurrent fever episodes in P2 over 5 weeks. Red dots represented increased temperatures during fever episodes and grey boxes represent the normal temperatures between flares. c, Representative sonographic image showing lymphadenopathy of P2. The diameters of right cervical lymph nodes were marked with dotted yellow line. d, Representative macroscopic image of dermatological manifestations in P1 during acute phase reaction, showing skin rashes (arrows). e, Whole blood cell counts (WBC), C-reactive protein (CRP) and serum amyloid A protein (SAA) were measured serially after the first evaluation of P1 and prednisone treatment (cyan shading) started at the age between 0-5. Horizontal dotted lines indicate age-specific high values for CRP, SAA or high and low values for WBC count. Red dots donate the abnormal values. f, C-reactive protein (CRP), serum ADA enzymatic activity and IL-6, TNF levels of unaffected controls (C, n=13), P1 during prednisone treatment (4 samples taken during flare and 5 samples taken during non-flare), P2 (4 samples taken during flare and 6 samples taken during non-flare), unaffected parents carrying heterozygous K377E mutation (K377E/WT, 1 sample from I-2 and 2 samples from II-2) or R390G mutation (R390G/WT, 3 samples from II-3). g, Sanger sequencing chromatograms of RIPK1 showed heterozygous base substitutions at p. K377E and p. R390G in whole blood genomic DNA from P1 and P2. h, WebLogo demonstrating conservation of human RIPK1 K377 and R390 across 42 vertebrate species. i-l, Cell death of SV40-immortalized human dermal fibroblasts (SV40-HDFs) isolated from three unaffected controls (C1-C3) and P2 were monitored by continuous imaging of SYTOX™ Orange staining. The SV40-HDFs were treated with T, S, Z as indicated ( i ). SV40-HDFs was treated with TZ with or without Nec-1s (10 μM) for 12 hours and immunoblotted with indicated antibodies ( j ). The TNFR signaling complex (TNF-RSC) were immunoprecipitated with Flag-TNF for 5 or 15 min and immunoblotted with indicated antibodies ( k ). NF-κB and MAPK activation of SV40-HDFs treated with TNF for 5, 15 or 30 min immunoblotted with indicated antibodies ( l ). m-p, RIPK1 -/- HT-29 cells were lentivirally complemented with HA-RIPK1 wild-type (WT), K377E, R390G and treated with T, S, Z as indicated. Cell viability was measured by CellTiter-Glo luminescent cell viability assay ( m ). The necroptosis markers ( n ), TNF-RSC ( o ) and the NF-κB and MAPK activation ( p ) were analyzed in the same way. T denotes 20 ng ml - TNF; S denotes 250 nM SM-164; Z denotes 25 μM <t>z-VAD-fmk.</t> Graphs show mean±SD. Statistical significance was determined by one-way ANOVA ( m ), **P < 0.01, ***P < 0.001.
    Z Vad Fmk, supplied by Selleck Chemicals, 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/z vad fmk/product/Selleck Chemicals
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    z vad fmk - by Bioz Stars, 2024-04
    86/100 stars

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    1) Product Images from "RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death"

    Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

    Journal: medRxiv

    doi: 10.1101/2024.03.28.24304774

    Compound heterozygous gain-of-function mutations in RIPK1 causes systemic autoinflammatory diseases. a , Pedigrees of a family with two patients carrying mutations at RIPK1 K377 and R390. Patient 1 (P1) and patient 2 (P2) were represented with filled symbols. b, Timeline of recurrent fever episodes in P2 over 5 weeks. Red dots represented increased temperatures during fever episodes and grey boxes represent the normal temperatures between flares. c, Representative sonographic image showing lymphadenopathy of P2. The diameters of right cervical lymph nodes were marked with dotted yellow line. d, Representative macroscopic image of dermatological manifestations in P1 during acute phase reaction, showing skin rashes (arrows). e, Whole blood cell counts (WBC), C-reactive protein (CRP) and serum amyloid A protein (SAA) were measured serially after the first evaluation of P1 and prednisone treatment (cyan shading) started at the age between 0-5. Horizontal dotted lines indicate age-specific high values for CRP, SAA or high and low values for WBC count. Red dots donate the abnormal values. f, C-reactive protein (CRP), serum ADA enzymatic activity and IL-6, TNF levels of unaffected controls (C, n=13), P1 during prednisone treatment (4 samples taken during flare and 5 samples taken during non-flare), P2 (4 samples taken during flare and 6 samples taken during non-flare), unaffected parents carrying heterozygous K377E mutation (K377E/WT, 1 sample from I-2 and 2 samples from II-2) or R390G mutation (R390G/WT, 3 samples from II-3). g, Sanger sequencing chromatograms of RIPK1 showed heterozygous base substitutions at p. K377E and p. R390G in whole blood genomic DNA from P1 and P2. h, WebLogo demonstrating conservation of human RIPK1 K377 and R390 across 42 vertebrate species. i-l, Cell death of SV40-immortalized human dermal fibroblasts (SV40-HDFs) isolated from three unaffected controls (C1-C3) and P2 were monitored by continuous imaging of SYTOX™ Orange staining. The SV40-HDFs were treated with T, S, Z as indicated ( i ). SV40-HDFs was treated with TZ with or without Nec-1s (10 μM) for 12 hours and immunoblotted with indicated antibodies ( j ). The TNFR signaling complex (TNF-RSC) were immunoprecipitated with Flag-TNF for 5 or 15 min and immunoblotted with indicated antibodies ( k ). NF-κB and MAPK activation of SV40-HDFs treated with TNF for 5, 15 or 30 min immunoblotted with indicated antibodies ( l ). m-p, RIPK1 -/- HT-29 cells were lentivirally complemented with HA-RIPK1 wild-type (WT), K377E, R390G and treated with T, S, Z as indicated. Cell viability was measured by CellTiter-Glo luminescent cell viability assay ( m ). The necroptosis markers ( n ), TNF-RSC ( o ) and the NF-κB and MAPK activation ( p ) were analyzed in the same way. T denotes 20 ng ml - TNF; S denotes 250 nM SM-164; Z denotes 25 μM z-VAD-fmk. Graphs show mean±SD. Statistical significance was determined by one-way ANOVA ( m ), **P < 0.01, ***P < 0.001.
    Figure Legend Snippet: Compound heterozygous gain-of-function mutations in RIPK1 causes systemic autoinflammatory diseases. a , Pedigrees of a family with two patients carrying mutations at RIPK1 K377 and R390. Patient 1 (P1) and patient 2 (P2) were represented with filled symbols. b, Timeline of recurrent fever episodes in P2 over 5 weeks. Red dots represented increased temperatures during fever episodes and grey boxes represent the normal temperatures between flares. c, Representative sonographic image showing lymphadenopathy of P2. The diameters of right cervical lymph nodes were marked with dotted yellow line. d, Representative macroscopic image of dermatological manifestations in P1 during acute phase reaction, showing skin rashes (arrows). e, Whole blood cell counts (WBC), C-reactive protein (CRP) and serum amyloid A protein (SAA) were measured serially after the first evaluation of P1 and prednisone treatment (cyan shading) started at the age between 0-5. Horizontal dotted lines indicate age-specific high values for CRP, SAA or high and low values for WBC count. Red dots donate the abnormal values. f, C-reactive protein (CRP), serum ADA enzymatic activity and IL-6, TNF levels of unaffected controls (C, n=13), P1 during prednisone treatment (4 samples taken during flare and 5 samples taken during non-flare), P2 (4 samples taken during flare and 6 samples taken during non-flare), unaffected parents carrying heterozygous K377E mutation (K377E/WT, 1 sample from I-2 and 2 samples from II-2) or R390G mutation (R390G/WT, 3 samples from II-3). g, Sanger sequencing chromatograms of RIPK1 showed heterozygous base substitutions at p. K377E and p. R390G in whole blood genomic DNA from P1 and P2. h, WebLogo demonstrating conservation of human RIPK1 K377 and R390 across 42 vertebrate species. i-l, Cell death of SV40-immortalized human dermal fibroblasts (SV40-HDFs) isolated from three unaffected controls (C1-C3) and P2 were monitored by continuous imaging of SYTOX™ Orange staining. The SV40-HDFs were treated with T, S, Z as indicated ( i ). SV40-HDFs was treated with TZ with or without Nec-1s (10 μM) for 12 hours and immunoblotted with indicated antibodies ( j ). The TNFR signaling complex (TNF-RSC) were immunoprecipitated with Flag-TNF for 5 or 15 min and immunoblotted with indicated antibodies ( k ). NF-κB and MAPK activation of SV40-HDFs treated with TNF for 5, 15 or 30 min immunoblotted with indicated antibodies ( l ). m-p, RIPK1 -/- HT-29 cells were lentivirally complemented with HA-RIPK1 wild-type (WT), K377E, R390G and treated with T, S, Z as indicated. Cell viability was measured by CellTiter-Glo luminescent cell viability assay ( m ). The necroptosis markers ( n ), TNF-RSC ( o ) and the NF-κB and MAPK activation ( p ) were analyzed in the same way. T denotes 20 ng ml - TNF; S denotes 250 nM SM-164; Z denotes 25 μM z-VAD-fmk. Graphs show mean±SD. Statistical significance was determined by one-way ANOVA ( m ), **P < 0.01, ***P < 0.001.

    Techniques Used: Activity Assay, Mutagenesis, Sequencing, Isolation, Imaging, Staining, Immunoprecipitation, Activation Assay, Cell Viability Assay

    RIPK1 mutations sensitize cells to TNF-induced cell death. a, SV40-HDFs isolated from three unaffected controls (C1-C3) and P2 were treated with T with or without S, Z and N as indicated. Cell death were monitored by imaging of SYTOX™ Orange staining. b-d, CellTiter-Glo luminescent cell viability detection of RIPK1 -/- HT-29 cells complemented with WT or mutated RIPK1 K377R, K377E, R390G, K377E/R390G under stimulation of TZ, TS, TSZ with or without N as indicated (b-c) . Expression levels of WT or mutated RIPK1 were determined with immunoblot (d) . e, mRNA levels of TNF , CXCL8 in complemented HT-29 cells treated with TNF for 6 hours were determined with qRT-PCR. f-g, HEK293T cells were transfected with plasmids encoding full length/intermediate domain of RIPK1 together with MIB2 or Ub as indicated. ID domain of RIPK1 or MIB2 were immunoprecipitated with anti-Flag dynabeads and immunoblotted with indicated antibodies. T denotes 20 ng ml - TNF; S denotes 250 nM SM-164; Z denotes 25 μM z-VAD-fmk; N denotes 10 μM Nec-1s. Graphs show mean±SD.
    Figure Legend Snippet: RIPK1 mutations sensitize cells to TNF-induced cell death. a, SV40-HDFs isolated from three unaffected controls (C1-C3) and P2 were treated with T with or without S, Z and N as indicated. Cell death were monitored by imaging of SYTOX™ Orange staining. b-d, CellTiter-Glo luminescent cell viability detection of RIPK1 -/- HT-29 cells complemented with WT or mutated RIPK1 K377R, K377E, R390G, K377E/R390G under stimulation of TZ, TS, TSZ with or without N as indicated (b-c) . Expression levels of WT or mutated RIPK1 were determined with immunoblot (d) . e, mRNA levels of TNF , CXCL8 in complemented HT-29 cells treated with TNF for 6 hours were determined with qRT-PCR. f-g, HEK293T cells were transfected with plasmids encoding full length/intermediate domain of RIPK1 together with MIB2 or Ub as indicated. ID domain of RIPK1 or MIB2 were immunoprecipitated with anti-Flag dynabeads and immunoblotted with indicated antibodies. T denotes 20 ng ml - TNF; S denotes 250 nM SM-164; Z denotes 25 μM z-VAD-fmk; N denotes 10 μM Nec-1s. Graphs show mean±SD.

    Techniques Used: Isolation, Imaging, Staining, Expressing, Western Blot, Quantitative RT-PCR, Transfection, Immunoprecipitation

    Strong activation of inflammatory signaling in myeloid cells carrying RIPK1 mutations. a , Integrated uniform manifold approximation and projection (UMAP) visualization of peripheral blood mononuclear cells (PBMCs) from three unaffected controls (C1, n=13925 cells; C2, n=11117 cells; C3, n=12573 cells) and two patients (P1, n=10918 cells; P2, n=8516 cells), marker-based annotation of 15 cell subtypes were colored by cluster identity. LDG, low-density granulocyte; pDC, plasmacytoid dendritic cell; Mo-DC, monocyte-derived dendritic cell; NK, natural killer cell; NKT, natural killer T cell; Eryth, erythrocyte; MK, megakaryocyte. b, Visualization of expressions of IL1B and CXCL8 (colored single cells) projecting PBMCs from unaffected controls (C1-C3) and two patients (P1 and P2) on UMAP plots. c, Boxplots of the NF-κB expression score (top panel) and IFN response score (bottom panel) of cell subtypes. d, Heatmap of representative genes downstream of NF-κB and type Ι IFN pathways in monocyte compartments. Gene names were listed in Extended Data Fig.4d. e, Dot plot showing the biological processes identified with un-regulated gene terms (FDR <0.05) in patients’ monocytes compared to that of unaffected controls using DAVID. The size of the circle indicates the gene count enriched in the pathway and the color represents pathway enrichment significance. f, Intracellular cytokine analysis of IL-1β, IL-6 and IFNγ in monocytes isolated from three unaffected (C1- C3) controls and P1. g, qRT-PCR analysis of IL1B, IL6, CXCL8 and TNF mRNA levels in primary monocytes isolated from unaffected controls (n=8-10) and two patients (n=4 from P1 and P2) with or without 10 ng ml - TNF for 6 hours. h, CellTiter-Glo luminescent cell viability detection of primary monocytes isolated from unaffected controls (n=12) and two patients (n=3 from P1 and P2) stimulated with T, S, Z for indicated times. i, Statistics of dead M1 macrophages stimulated with TS or TSZ for 24 hours by imaging of propidium iodide (PI). M1 macrophages were differentiated from primary monocytes of unaffected controls (n=6) and two patients (n=4 from P1 and P2). Representative images were presented in Extended Data Fig.3e. j, Volcano plot displaying the differentially expressed genes in monocyte compartments between three unaffected controls (C1-C3) and two patients (P1 and P2). Representative suppressive genes of cell death were labeled with red dots and gene names. k, Clusters in the monocyte compartments showing the expression levels of TNFAIP3 and BCL2A1 in three unaffected controls (C1-C3) and two patients (P1 and P2). l, Schematic diagram showing serum cytokine microarray analysis of continuous P2’s serum samples before (PS1 and PS2) or after (PS3, flare; PS4 and PS5, non-flare) the onset of recurrent fever. m, PCA analysis of sample variance determined by semi-quantitative 174 cytokines antibody array on serum samples of C1-C3 from three unaffected controls and PS1-PS5 from P2. n, The integrated relative MFI plots of P2’s serum cytokine levels. 28 cytokines peaked in PS3 were enrich in group 1, 24 cytokines higher than control were enriched in group 2. o, Boxplots exhibiting mRNA expression scores of cytokines of group 1 (left panel) and group 2 (right panel) in indicated cell compartments. Group 1 and group 2 cytokine panels were clustered with the serum cytokine microarray analysis in Extended Data Fig.4. T denotes 10 ng ml - TNF; S denotes 250 nM SM164; Z denotes 25 μM z-VAD-fmk. All histogram graphs show mean±SD. Statistical significance was determined by two-tailed unpaired t-test ( c, g, i, l ) or one-way ANOVA ( h ), ns, P > 0.05, * P <0.05, ** P < 0.01, *** P < 0.001.
    Figure Legend Snippet: Strong activation of inflammatory signaling in myeloid cells carrying RIPK1 mutations. a , Integrated uniform manifold approximation and projection (UMAP) visualization of peripheral blood mononuclear cells (PBMCs) from three unaffected controls (C1, n=13925 cells; C2, n=11117 cells; C3, n=12573 cells) and two patients (P1, n=10918 cells; P2, n=8516 cells), marker-based annotation of 15 cell subtypes were colored by cluster identity. LDG, low-density granulocyte; pDC, plasmacytoid dendritic cell; Mo-DC, monocyte-derived dendritic cell; NK, natural killer cell; NKT, natural killer T cell; Eryth, erythrocyte; MK, megakaryocyte. b, Visualization of expressions of IL1B and CXCL8 (colored single cells) projecting PBMCs from unaffected controls (C1-C3) and two patients (P1 and P2) on UMAP plots. c, Boxplots of the NF-κB expression score (top panel) and IFN response score (bottom panel) of cell subtypes. d, Heatmap of representative genes downstream of NF-κB and type Ι IFN pathways in monocyte compartments. Gene names were listed in Extended Data Fig.4d. e, Dot plot showing the biological processes identified with un-regulated gene terms (FDR <0.05) in patients’ monocytes compared to that of unaffected controls using DAVID. The size of the circle indicates the gene count enriched in the pathway and the color represents pathway enrichment significance. f, Intracellular cytokine analysis of IL-1β, IL-6 and IFNγ in monocytes isolated from three unaffected (C1- C3) controls and P1. g, qRT-PCR analysis of IL1B, IL6, CXCL8 and TNF mRNA levels in primary monocytes isolated from unaffected controls (n=8-10) and two patients (n=4 from P1 and P2) with or without 10 ng ml - TNF for 6 hours. h, CellTiter-Glo luminescent cell viability detection of primary monocytes isolated from unaffected controls (n=12) and two patients (n=3 from P1 and P2) stimulated with T, S, Z for indicated times. i, Statistics of dead M1 macrophages stimulated with TS or TSZ for 24 hours by imaging of propidium iodide (PI). M1 macrophages were differentiated from primary monocytes of unaffected controls (n=6) and two patients (n=4 from P1 and P2). Representative images were presented in Extended Data Fig.3e. j, Volcano plot displaying the differentially expressed genes in monocyte compartments between three unaffected controls (C1-C3) and two patients (P1 and P2). Representative suppressive genes of cell death were labeled with red dots and gene names. k, Clusters in the monocyte compartments showing the expression levels of TNFAIP3 and BCL2A1 in three unaffected controls (C1-C3) and two patients (P1 and P2). l, Schematic diagram showing serum cytokine microarray analysis of continuous P2’s serum samples before (PS1 and PS2) or after (PS3, flare; PS4 and PS5, non-flare) the onset of recurrent fever. m, PCA analysis of sample variance determined by semi-quantitative 174 cytokines antibody array on serum samples of C1-C3 from three unaffected controls and PS1-PS5 from P2. n, The integrated relative MFI plots of P2’s serum cytokine levels. 28 cytokines peaked in PS3 were enrich in group 1, 24 cytokines higher than control were enriched in group 2. o, Boxplots exhibiting mRNA expression scores of cytokines of group 1 (left panel) and group 2 (right panel) in indicated cell compartments. Group 1 and group 2 cytokine panels were clustered with the serum cytokine microarray analysis in Extended Data Fig.4. T denotes 10 ng ml - TNF; S denotes 250 nM SM164; Z denotes 25 μM z-VAD-fmk. All histogram graphs show mean±SD. Statistical significance was determined by two-tailed unpaired t-test ( c, g, i, l ) or one-way ANOVA ( h ), ns, P > 0.05, * P <0.05, ** P < 0.01, *** P < 0.001.

    Techniques Used: Activation Assay, Marker, Derivative Assay, Expressing, Isolation, Quantitative RT-PCR, Imaging, Labeling, Microarray, Ab Array, Two Tailed Test

    Increased cell death and CD4/CD8 ratio in T cells caused by RIPK1 activation. a , Average expression values (arcsinh transformed) of cl. Casp-3 across identified peripheral immune cell lineages in PBMCs freshly isolated from four unaffected controls (C) and two patients (P, 1 sample from P1 and 2 samples from P2) by mass cytometry by time-of-flight (CyTOF) analysis. b-e, Representative contour plots of cl. Casp-3 ( b-c ) and histograms of total RIPK1, pRIPK1(S166) ( d-e ) levels in CD4 + T and CD8 + T cells in freshly isolated PBMCs from unaffected controls (n=10-12) and patients (n=4 for P1 and P2) (left panel). Summary histograms of cl. Casp-3 and pRIPK1(S166) (right panel) were shown. f, Immunoblotting analysis of primary CD3 + T cells isolated from two unaffected controls (C1 and C2) and P1 treated with T and S with or without Z, N for 6 hours. Antibodies as indicated were immunoblotted to show the activation of apoptosis or necroptosis. g, CellTiter-Glo luminescent cell viability detection of primary CD3 + T cells isolated from three unaffected controls (C1-C3), two patients (P1 and P2) and their parents treated with TS or TSZ for indicated times, n=4. h, Representative histogram and statistical analysis of surface CD69 expression on CD3 + T cells from unaffected controls (n=7) and two patients (n=3 for P1 and P2). i, Serum IFNγ, sCD25, sCD95 and CD95L of unaffected controls (C, n=10), two patients (P1, n=5-7; P2, n=4-8) and their parents (n=6-7) as well as two CRIA syndrome patients (n=6) determined by ELISA. j, CD4/CD8 ratio in T cells, percentages and absolute counts of CD3 + T, CD4 + T, CD8 + T cells in whole blood sample of P1 (red dots) and P2 (blue dots) measured using immunophenotyping. k, In vitro expansion of primary T cells isolated from unaffected controls (n=7) and patients (n=3 for P1 and P2) as indicated. Cell numbers were counted every two days post anti-CD3/CD28 dynabeads stimulation. l, Proliferation of CD4 + T and CD8 + T cells isolated from three unaffected controls (C1-C3) and P1 were analyzed using CFSE staining upon stimulation with anti-CD3/CD28 dynabeads for 96 hours. m, Representative histograms of surface CD95 expression on CD3 + T cells analyzed with flow cytometry after culturing in vitro for 24 hours. n, Representative contour plots depicting the percentages of CD4 + T, CD8 + T, double negative T (DNT) and double positive T (DPT) in CD3 + T cells gated from PBMCs of P2 culturing in vitro for 24 hours. Histograms displaying cl. Casp-3 and RIPK1 expression levels in CD8 hi T and CD8 lo T cells. o, Representative contour plots and summary histograms showing percentages of CD4 + T, CD8 + T and CD8 hi T cells and CD4/CD8 ratio in CD3 + T cells isolated from P2 cultured for indicated times. p-q, Representative contour plots showing percentages of CD4 + T, CD8 + T in CD3 + T cells of P2 with (TurboGFP + ) or without (TurboGFP - ) restoration using CRISPR/Cas9-mediated in situ knock-in of WT RIPK1-P2A-TurboGFP under stimulation of T with or without S, Z for 6 hours ( p ). CD4/CD8 ratio in CD3 + T cells of unaffected controls (n=7) and two patients (n=3 for P1 and P2) were quantified ( q ). r-s, Representative contour plots ( r ) and statistical analysis ( s ) showing percentages of CD4 + T, CD8 + T, DNT and DPT cell in CD3 + T cells isolated from unaffected control (n=7) and two patients (n=9 for P1 and P2) under the stimulation of anti-CD3 antibody (5 μg ml - ) for 72 hours. t, Statistical analysis of fold change of CD4 /CD8 ratio in CD3 + T cells isolated from two patients (n=4 for P1 and P2) under stimulation of anti-CD3 antibody (5 μg ml - ) with or without 250 nM Ponatinib or 10 μM Nec-1s for 24 hours. For representative contour plots, see Extended Data Fig. 6h. T denotes 10 ng ml - TNF; S denotes 250 nM SM164; Z denotes 25 μM z-VAD-fmk; N denotes 10 μM Nec-1s. All histogram graphs show mean±SD. Statistical significance was determined by one-way ANOVA ( g) or two-tailed unpaired t-test in the rest, * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
    Figure Legend Snippet: Increased cell death and CD4/CD8 ratio in T cells caused by RIPK1 activation. a , Average expression values (arcsinh transformed) of cl. Casp-3 across identified peripheral immune cell lineages in PBMCs freshly isolated from four unaffected controls (C) and two patients (P, 1 sample from P1 and 2 samples from P2) by mass cytometry by time-of-flight (CyTOF) analysis. b-e, Representative contour plots of cl. Casp-3 ( b-c ) and histograms of total RIPK1, pRIPK1(S166) ( d-e ) levels in CD4 + T and CD8 + T cells in freshly isolated PBMCs from unaffected controls (n=10-12) and patients (n=4 for P1 and P2) (left panel). Summary histograms of cl. Casp-3 and pRIPK1(S166) (right panel) were shown. f, Immunoblotting analysis of primary CD3 + T cells isolated from two unaffected controls (C1 and C2) and P1 treated with T and S with or without Z, N for 6 hours. Antibodies as indicated were immunoblotted to show the activation of apoptosis or necroptosis. g, CellTiter-Glo luminescent cell viability detection of primary CD3 + T cells isolated from three unaffected controls (C1-C3), two patients (P1 and P2) and their parents treated with TS or TSZ for indicated times, n=4. h, Representative histogram and statistical analysis of surface CD69 expression on CD3 + T cells from unaffected controls (n=7) and two patients (n=3 for P1 and P2). i, Serum IFNγ, sCD25, sCD95 and CD95L of unaffected controls (C, n=10), two patients (P1, n=5-7; P2, n=4-8) and their parents (n=6-7) as well as two CRIA syndrome patients (n=6) determined by ELISA. j, CD4/CD8 ratio in T cells, percentages and absolute counts of CD3 + T, CD4 + T, CD8 + T cells in whole blood sample of P1 (red dots) and P2 (blue dots) measured using immunophenotyping. k, In vitro expansion of primary T cells isolated from unaffected controls (n=7) and patients (n=3 for P1 and P2) as indicated. Cell numbers were counted every two days post anti-CD3/CD28 dynabeads stimulation. l, Proliferation of CD4 + T and CD8 + T cells isolated from three unaffected controls (C1-C3) and P1 were analyzed using CFSE staining upon stimulation with anti-CD3/CD28 dynabeads for 96 hours. m, Representative histograms of surface CD95 expression on CD3 + T cells analyzed with flow cytometry after culturing in vitro for 24 hours. n, Representative contour plots depicting the percentages of CD4 + T, CD8 + T, double negative T (DNT) and double positive T (DPT) in CD3 + T cells gated from PBMCs of P2 culturing in vitro for 24 hours. Histograms displaying cl. Casp-3 and RIPK1 expression levels in CD8 hi T and CD8 lo T cells. o, Representative contour plots and summary histograms showing percentages of CD4 + T, CD8 + T and CD8 hi T cells and CD4/CD8 ratio in CD3 + T cells isolated from P2 cultured for indicated times. p-q, Representative contour plots showing percentages of CD4 + T, CD8 + T in CD3 + T cells of P2 with (TurboGFP + ) or without (TurboGFP - ) restoration using CRISPR/Cas9-mediated in situ knock-in of WT RIPK1-P2A-TurboGFP under stimulation of T with or without S, Z for 6 hours ( p ). CD4/CD8 ratio in CD3 + T cells of unaffected controls (n=7) and two patients (n=3 for P1 and P2) were quantified ( q ). r-s, Representative contour plots ( r ) and statistical analysis ( s ) showing percentages of CD4 + T, CD8 + T, DNT and DPT cell in CD3 + T cells isolated from unaffected control (n=7) and two patients (n=9 for P1 and P2) under the stimulation of anti-CD3 antibody (5 μg ml - ) for 72 hours. t, Statistical analysis of fold change of CD4 /CD8 ratio in CD3 + T cells isolated from two patients (n=4 for P1 and P2) under stimulation of anti-CD3 antibody (5 μg ml - ) with or without 250 nM Ponatinib or 10 μM Nec-1s for 24 hours. For representative contour plots, see Extended Data Fig. 6h. T denotes 10 ng ml - TNF; S denotes 250 nM SM164; Z denotes 25 μM z-VAD-fmk; N denotes 10 μM Nec-1s. All histogram graphs show mean±SD. Statistical significance was determined by one-way ANOVA ( g) or two-tailed unpaired t-test in the rest, * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

    Techniques Used: Activation Assay, Expressing, Transformation Assay, Isolation, Mass Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, In Vitro, Staining, Flow Cytometry, Cell Culture, CRISPR, In Situ, Knock-In, Two Tailed Test

    RIPK1-mediated cell death leads to CD8 decrease in T cells. a, Percentages and absolute counts of total lymphocytes, γδ T cells, NKT cells and B cells from whole blood of P1 (red dots) and P2 (blue dots) measured by immunophenotyping. b, Representative contour plots showing percentages of CD8 hi T, CD8 lo T, CD4 + T, DNT and DPT cells in CD3 + T cells isolated from unaffected control and P1 under the stimulation of TS, TSZ and cytokines (combination of IL-1β (10 ng ml - ), IL-6 (100 ng ml - ), IFNα (100 ng ml - ) and IL-8 (100 ng ml - )) as indicated for 6 hours. c- d, Representative contour plots ( c ) and summary histogram ( d ) showing percentages of CD8 hi T, CD8 lo T, CD4 + T, DNT and DPT cells in CD3 + T cells isolated from P2 under the stimulation of TS, TSZ, TSN and TSZN as indicated for 6 hours. e, Schematic of CRISPR/Cas9-mediated in situ knock-in of WT RIPK1 into patients’ RIPK1 locus. f, Representative contour plots of Turbo-GFP + cells percentage indicating WT RIPK1 knockin efficiency. g, Statistical analysis of CD8 + T cell proportions in CD3 + T cells from unaffected controls (n=3-6) and two patients (n=3 for P1 and P2) under the stimulation of TS, TSZ as well as RSL3 (1 μM) and STS (100 nM) for indicated times. h, Representative contour plots showing percentages of CD8 + T and CD4 + T cells in CD3 + T cells isolated from P2 under the stimulation of anti-CD3 antibody (5 μg/ml) with or without Ponatinib (250 nM) and Nec-1s (10 μM) for 24 hours. T denotes 20 ng ml - TNF; S denotes 250 nM SM-164; Z denotes 25 μM z-VAD-fmk; N denotes 10 μM Nec-1s. Graphs show mean±SD.
    Figure Legend Snippet: RIPK1-mediated cell death leads to CD8 decrease in T cells. a, Percentages and absolute counts of total lymphocytes, γδ T cells, NKT cells and B cells from whole blood of P1 (red dots) and P2 (blue dots) measured by immunophenotyping. b, Representative contour plots showing percentages of CD8 hi T, CD8 lo T, CD4 + T, DNT and DPT cells in CD3 + T cells isolated from unaffected control and P1 under the stimulation of TS, TSZ and cytokines (combination of IL-1β (10 ng ml - ), IL-6 (100 ng ml - ), IFNα (100 ng ml - ) and IL-8 (100 ng ml - )) as indicated for 6 hours. c- d, Representative contour plots ( c ) and summary histogram ( d ) showing percentages of CD8 hi T, CD8 lo T, CD4 + T, DNT and DPT cells in CD3 + T cells isolated from P2 under the stimulation of TS, TSZ, TSN and TSZN as indicated for 6 hours. e, Schematic of CRISPR/Cas9-mediated in situ knock-in of WT RIPK1 into patients’ RIPK1 locus. f, Representative contour plots of Turbo-GFP + cells percentage indicating WT RIPK1 knockin efficiency. g, Statistical analysis of CD8 + T cell proportions in CD3 + T cells from unaffected controls (n=3-6) and two patients (n=3 for P1 and P2) under the stimulation of TS, TSZ as well as RSL3 (1 μM) and STS (100 nM) for indicated times. h, Representative contour plots showing percentages of CD8 + T and CD4 + T cells in CD3 + T cells isolated from P2 under the stimulation of anti-CD3 antibody (5 μg/ml) with or without Ponatinib (250 nM) and Nec-1s (10 μM) for 24 hours. T denotes 20 ng ml - TNF; S denotes 250 nM SM-164; Z denotes 25 μM z-VAD-fmk; N denotes 10 μM Nec-1s. Graphs show mean±SD.

    Techniques Used: Isolation, CRISPR, In Situ, Knock-In

    RIPK1 activation causes inflammation through T-Mono axis. a , Schematic diagram of T cell-MDM co-culture system. b-c, qRT-PCR analysis of TNF, IL1B and IL6 mRNA levels in GM-CSF-activated monocytes derived macrophages (MDMs) co-cultured with or without T cells for 6 hours. GM-CSF-activated MDMs generated from unaffected control 1 (C1) (left panel) or unaffected control 3 (C3) (right panel) ( b ) or P1 ( c ) were co-cultured with donor-matched T cells as well as T cells from other unaffected controls (C2 and C4) or patients as indicated. d, Dot plot of IFNG, TNF in cell subtypes found in scRNA-seq of PBMCs from three unaffected controls and two patients. The size of the circle indicates the percentage of cells positive for gene expression and the color represents the average expression level. e, Representative histograms and statistical analysis of TNF and IFNγ expression in CD8 + T cells in PBMCs isolated from unaffected controls (n=6) and patients (n=3 for P1 and P2) treated with 10 ng ml - TNF (T) with or without 250 nM SM-164 (S), 25 μM z-VAD-fmk (Z) for 24 hours. f, T cells isolated from P2 were co-cultured with MDMs generated from unaffected control in the presence of 10 μg ml - adalimumab, 10 μg ml - emapalumab alone or in combination for 6 hours. TNF, IL1B and IL6 mRNA levels of MDMs were analyzed by qRT-PCR. g, Experimental timeline for generation of HSCs-transplanted mice with conditional overexpression of human WT or mutated RIPK1 K377E, R390G or D324V in CD8α + T cell- or myeloid cell-specific lineage. h-i, ELISA measurement of serum Tnf and Il6 levels in mice specifically overexpressing WT or mutated hRIPK1 in CD8α + T cells ( h ) or myeloid cells ( i ). j-m, Pedigrees of two families with CRIA patients (CRIA-1 and CRIA-2) ( j ). Patients were represented with filled symbols. Summary histograms of CD4/CD8 ratio in CD3 + T cells of unaffected controls (n=12), CRIA-1 (n=7) and CRIA-2 (n=5) were shown ( k). Representative contour plots of cl. Casp-3 ( l ) and histograms of pRIPK1(S166) ( m ) protein levels in CD8 + T cells in freshly isolated PBMCs from CRIA patients. n-o, Whole blood cell counts (WBC), cell counts and proportions of neutrophil (NE#-NE%) and lymphocyte (LYMPH# -LYMPH%) ( n) , Serum C-reactive protein (CRP), TNF and IL-6 ( o ) of P1 (red dots) and P2 (blue dots) measured before and after tocilizumab or adalimumab treatments. The grey area represents the range of unaffected controls. Dots represent each sample and graphs show mean±SD. Statistical significance was determined by two-tailed unpaired t-test, * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
    Figure Legend Snippet: RIPK1 activation causes inflammation through T-Mono axis. a , Schematic diagram of T cell-MDM co-culture system. b-c, qRT-PCR analysis of TNF, IL1B and IL6 mRNA levels in GM-CSF-activated monocytes derived macrophages (MDMs) co-cultured with or without T cells for 6 hours. GM-CSF-activated MDMs generated from unaffected control 1 (C1) (left panel) or unaffected control 3 (C3) (right panel) ( b ) or P1 ( c ) were co-cultured with donor-matched T cells as well as T cells from other unaffected controls (C2 and C4) or patients as indicated. d, Dot plot of IFNG, TNF in cell subtypes found in scRNA-seq of PBMCs from three unaffected controls and two patients. The size of the circle indicates the percentage of cells positive for gene expression and the color represents the average expression level. e, Representative histograms and statistical analysis of TNF and IFNγ expression in CD8 + T cells in PBMCs isolated from unaffected controls (n=6) and patients (n=3 for P1 and P2) treated with 10 ng ml - TNF (T) with or without 250 nM SM-164 (S), 25 μM z-VAD-fmk (Z) for 24 hours. f, T cells isolated from P2 were co-cultured with MDMs generated from unaffected control in the presence of 10 μg ml - adalimumab, 10 μg ml - emapalumab alone or in combination for 6 hours. TNF, IL1B and IL6 mRNA levels of MDMs were analyzed by qRT-PCR. g, Experimental timeline for generation of HSCs-transplanted mice with conditional overexpression of human WT or mutated RIPK1 K377E, R390G or D324V in CD8α + T cell- or myeloid cell-specific lineage. h-i, ELISA measurement of serum Tnf and Il6 levels in mice specifically overexpressing WT or mutated hRIPK1 in CD8α + T cells ( h ) or myeloid cells ( i ). j-m, Pedigrees of two families with CRIA patients (CRIA-1 and CRIA-2) ( j ). Patients were represented with filled symbols. Summary histograms of CD4/CD8 ratio in CD3 + T cells of unaffected controls (n=12), CRIA-1 (n=7) and CRIA-2 (n=5) were shown ( k). Representative contour plots of cl. Casp-3 ( l ) and histograms of pRIPK1(S166) ( m ) protein levels in CD8 + T cells in freshly isolated PBMCs from CRIA patients. n-o, Whole blood cell counts (WBC), cell counts and proportions of neutrophil (NE#-NE%) and lymphocyte (LYMPH# -LYMPH%) ( n) , Serum C-reactive protein (CRP), TNF and IL-6 ( o ) of P1 (red dots) and P2 (blue dots) measured before and after tocilizumab or adalimumab treatments. The grey area represents the range of unaffected controls. Dots represent each sample and graphs show mean±SD. Statistical significance was determined by two-tailed unpaired t-test, * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

    Techniques Used: Activation Assay, Co-Culture Assay, Quantitative RT-PCR, Derivative Assay, Cell Culture, Generated, Expressing, Isolation, Over Expression, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    z vad fmk  (Selleck Chemicals)


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    Phendione induces apoptosis in human PDAC cells. a The human PDAC cell line MIA PaCA-2 was incubated with up to 10 µM phendione for 24 h and 48 h. Apoptosis was determined by flow cytometry for annexin-V/PI ( n = 3). Data were statistically analyzed using two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). b MIA PaCA-2 were treated with increasing doses of phendione up to 10 µM and collected after 24 h. The total and cleaved forms of the apoptosis marker caspase-3 were detected by immunoblot. HSP90 is the loading control; n = 2. c Apoptosis was measured by flow cytometry of MIA PaCa-2 cells that were exposed for 24 h to 3 µM phendione and 50 µM of the caspase inhibitor <t>Z-VAD-FMK</t> ( n = 3). Data were statistically analyzed using two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). d MIA PaCA-2 cells were cultured with 1 µM, 3 µM, 5 µM, and 10 µM phendione for 24 h. Immunoblot was performed for ATM, p-ATM (S1981), p-KAP1 (S824), AKT, p-AKT (S473), ERK and p-ERK (T202/Y204). Vinculin is used as a loading control; n = 3. e MIA PaCA-2 cells were stimulated with increasing concentrations of phendione (1–10 µM). Immunoblot analyses of whole cell lysates were performed to detect PR130. HSP90 served as loading control; n = 3. Quantification was done by normalizing the PR130 signals to those of vinculin. f Immunoblot of MIA PaCA-2 cells, which were treated with phendione and the proteasomal inhibitor lactacystin was performed to detect PR130. Quantification was done by normalizing the PR130 signals to those of vinculin which was used as loading control; n = 3
    Z Vad Fmk, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The protein phosphatase-2A subunit PR130 is involved in the formation of cytotoxic protein aggregates in pancreatic ductal adenocarcinoma cells"

    Article Title: The protein phosphatase-2A subunit PR130 is involved in the formation of cytotoxic protein aggregates in pancreatic ductal adenocarcinoma cells

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-024-01597-8

    Phendione induces apoptosis in human PDAC cells. a The human PDAC cell line MIA PaCA-2 was incubated with up to 10 µM phendione for 24 h and 48 h. Apoptosis was determined by flow cytometry for annexin-V/PI ( n = 3). Data were statistically analyzed using two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). b MIA PaCA-2 were treated with increasing doses of phendione up to 10 µM and collected after 24 h. The total and cleaved forms of the apoptosis marker caspase-3 were detected by immunoblot. HSP90 is the loading control; n = 2. c Apoptosis was measured by flow cytometry of MIA PaCa-2 cells that were exposed for 24 h to 3 µM phendione and 50 µM of the caspase inhibitor Z-VAD-FMK ( n = 3). Data were statistically analyzed using two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). d MIA PaCA-2 cells were cultured with 1 µM, 3 µM, 5 µM, and 10 µM phendione for 24 h. Immunoblot was performed for ATM, p-ATM (S1981), p-KAP1 (S824), AKT, p-AKT (S473), ERK and p-ERK (T202/Y204). Vinculin is used as a loading control; n = 3. e MIA PaCA-2 cells were stimulated with increasing concentrations of phendione (1–10 µM). Immunoblot analyses of whole cell lysates were performed to detect PR130. HSP90 served as loading control; n = 3. Quantification was done by normalizing the PR130 signals to those of vinculin. f Immunoblot of MIA PaCA-2 cells, which were treated with phendione and the proteasomal inhibitor lactacystin was performed to detect PR130. Quantification was done by normalizing the PR130 signals to those of vinculin which was used as loading control; n = 3
    Figure Legend Snippet: Phendione induces apoptosis in human PDAC cells. a The human PDAC cell line MIA PaCA-2 was incubated with up to 10 µM phendione for 24 h and 48 h. Apoptosis was determined by flow cytometry for annexin-V/PI ( n = 3). Data were statistically analyzed using two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). b MIA PaCA-2 were treated with increasing doses of phendione up to 10 µM and collected after 24 h. The total and cleaved forms of the apoptosis marker caspase-3 were detected by immunoblot. HSP90 is the loading control; n = 2. c Apoptosis was measured by flow cytometry of MIA PaCa-2 cells that were exposed for 24 h to 3 µM phendione and 50 µM of the caspase inhibitor Z-VAD-FMK ( n = 3). Data were statistically analyzed using two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). d MIA PaCA-2 cells were cultured with 1 µM, 3 µM, 5 µM, and 10 µM phendione for 24 h. Immunoblot was performed for ATM, p-ATM (S1981), p-KAP1 (S824), AKT, p-AKT (S473), ERK and p-ERK (T202/Y204). Vinculin is used as a loading control; n = 3. e MIA PaCA-2 cells were stimulated with increasing concentrations of phendione (1–10 µM). Immunoblot analyses of whole cell lysates were performed to detect PR130. HSP90 served as loading control; n = 3. Quantification was done by normalizing the PR130 signals to those of vinculin. f Immunoblot of MIA PaCA-2 cells, which were treated with phendione and the proteasomal inhibitor lactacystin was performed to detect PR130. Quantification was done by normalizing the PR130 signals to those of vinculin which was used as loading control; n = 3

    Techniques Used: Incubation, Flow Cytometry, Marker, Western Blot, Cell Culture

    z vad fmk  (Selleck Chemicals)


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    Selleck Chemicals z vad fmk
    Z Vad Fmk, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    z vad fmk s7023  (Selleck Chemicals)


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    Z Vad Fmk S7023, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    z vad fmk  (Selleck Chemicals)


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    Z Vad Fmk, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    z vad fmk z vad  (Selleck Chemicals)


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    Juglone induces cell death phenotypic changes in GBM cells. A LN229 and T98G cells were pretreated with inhibitors such as Fer-1 (4 μM), Lip-1 (0.1 μM), Nec-1 (10 μM), <t>Z-VAD</t> (25 μM), CQ-1(10 μM) or Mdivi(20 μM) for 1 h and then co-treated with juglone for 24 h. The cell viability was determined through a CCK-8 assay. B , C LN229 and T98G cells were treated with different concentrations of juglone for 24 h. The ROS levels of each group of cells were detected and quantitatively analyzed through flow cytometry. D , E The levels of GSH and MDA in LN229 and T98G cells were measured under the effect of different concentrations of juglone. F , G After 24 h of treatment with different concentrations of juglone, the 4HNE level in LN229 and T98G cells was detected and quantitatively analyzed through immunofluorescence. Fer-1 ferrostatin-1, Lip-1 liproxstatin-1, Nec necrostatin-1, Z-VAD <t>Z-VAD-FMK,</t> CQ-1 chloroquine, Mdivi Mdivi-1, GSH glutathione, MDA malonaldehyde; *p < 0.05; **p < 0.01; ***p < 0.001; ns, no statistical significance
    Z Vad Fmk Z Vad, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Juglone induces ferroptosis in glioblastoma cells by inhibiting the Nrf2-GPX4 axis through the phosphorylation of p38MAPK"

    Article Title: Juglone induces ferroptosis in glioblastoma cells by inhibiting the Nrf2-GPX4 axis through the phosphorylation of p38MAPK

    Journal: Chinese Medicine

    doi: 10.1186/s13020-024-00920-2

    Juglone induces cell death phenotypic changes in GBM cells. A LN229 and T98G cells were pretreated with inhibitors such as Fer-1 (4 μM), Lip-1 (0.1 μM), Nec-1 (10 μM), Z-VAD (25 μM), CQ-1(10 μM) or Mdivi(20 μM) for 1 h and then co-treated with juglone for 24 h. The cell viability was determined through a CCK-8 assay. B , C LN229 and T98G cells were treated with different concentrations of juglone for 24 h. The ROS levels of each group of cells were detected and quantitatively analyzed through flow cytometry. D , E The levels of GSH and MDA in LN229 and T98G cells were measured under the effect of different concentrations of juglone. F , G After 24 h of treatment with different concentrations of juglone, the 4HNE level in LN229 and T98G cells was detected and quantitatively analyzed through immunofluorescence. Fer-1 ferrostatin-1, Lip-1 liproxstatin-1, Nec necrostatin-1, Z-VAD Z-VAD-FMK, CQ-1 chloroquine, Mdivi Mdivi-1, GSH glutathione, MDA malonaldehyde; *p < 0.05; **p < 0.01; ***p < 0.001; ns, no statistical significance
    Figure Legend Snippet: Juglone induces cell death phenotypic changes in GBM cells. A LN229 and T98G cells were pretreated with inhibitors such as Fer-1 (4 μM), Lip-1 (0.1 μM), Nec-1 (10 μM), Z-VAD (25 μM), CQ-1(10 μM) or Mdivi(20 μM) for 1 h and then co-treated with juglone for 24 h. The cell viability was determined through a CCK-8 assay. B , C LN229 and T98G cells were treated with different concentrations of juglone for 24 h. The ROS levels of each group of cells were detected and quantitatively analyzed through flow cytometry. D , E The levels of GSH and MDA in LN229 and T98G cells were measured under the effect of different concentrations of juglone. F , G After 24 h of treatment with different concentrations of juglone, the 4HNE level in LN229 and T98G cells was detected and quantitatively analyzed through immunofluorescence. Fer-1 ferrostatin-1, Lip-1 liproxstatin-1, Nec necrostatin-1, Z-VAD Z-VAD-FMK, CQ-1 chloroquine, Mdivi Mdivi-1, GSH glutathione, MDA malonaldehyde; *p < 0.05; **p < 0.01; ***p < 0.001; ns, no statistical significance

    Techniques Used: CCK-8 Assay, Flow Cytometry, Immunofluorescence

    z vad fmk  (Selleck Chemicals)


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    scRNA-seq identified lethal levels of ROS in DTEPs after BET inhibition (A) Comparison of ROS pathway gene signature module scores on parental, DTP, DTEP-UN, and DTEP-TR. (B and C) Flow cytometry-based quantification of intracellular ROS level (mean fluorescent intensity) in on parental, DTP, DTEP-UN, and DTEP-TR in both PC9 (B) and A375 (C) cells. (Samples collected at 72 h, NEO2734: 0.25 μM). (D and E) Relative cell viability of DTEPs exposure to BET inhibitors with or without NAC, ferrostatin, and <t>Z-VAD-FMK</t> (NEO2734: 0.25 μM; NAC: 2.5 mM; ferrostatin: 2.5 μM; Z-VAD-FMK: 10 μM). Error bars in (B)–(E) represent mean ± SD, n = 3 independent experiments. Statistical significance was calculated by 2-tailed Student’s t test (ns, not significant; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001).
    Z Vad Fmk, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Targeting of vulnerabilities of drug-tolerant persisters identified through functional genetics delays tumor relapse"

    Article Title: Targeting of vulnerabilities of drug-tolerant persisters identified through functional genetics delays tumor relapse

    Journal: Cell Reports Medicine

    doi: 10.1016/j.xcrm.2024.101471

    scRNA-seq identified lethal levels of ROS in DTEPs after BET inhibition (A) Comparison of ROS pathway gene signature module scores on parental, DTP, DTEP-UN, and DTEP-TR. (B and C) Flow cytometry-based quantification of intracellular ROS level (mean fluorescent intensity) in on parental, DTP, DTEP-UN, and DTEP-TR in both PC9 (B) and A375 (C) cells. (Samples collected at 72 h, NEO2734: 0.25 μM). (D and E) Relative cell viability of DTEPs exposure to BET inhibitors with or without NAC, ferrostatin, and Z-VAD-FMK (NEO2734: 0.25 μM; NAC: 2.5 mM; ferrostatin: 2.5 μM; Z-VAD-FMK: 10 μM). Error bars in (B)–(E) represent mean ± SD, n = 3 independent experiments. Statistical significance was calculated by 2-tailed Student’s t test (ns, not significant; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001).
    Figure Legend Snippet: scRNA-seq identified lethal levels of ROS in DTEPs after BET inhibition (A) Comparison of ROS pathway gene signature module scores on parental, DTP, DTEP-UN, and DTEP-TR. (B and C) Flow cytometry-based quantification of intracellular ROS level (mean fluorescent intensity) in on parental, DTP, DTEP-UN, and DTEP-TR in both PC9 (B) and A375 (C) cells. (Samples collected at 72 h, NEO2734: 0.25 μM). (D and E) Relative cell viability of DTEPs exposure to BET inhibitors with or without NAC, ferrostatin, and Z-VAD-FMK (NEO2734: 0.25 μM; NAC: 2.5 mM; ferrostatin: 2.5 μM; Z-VAD-FMK: 10 μM). Error bars in (B)–(E) represent mean ± SD, n = 3 independent experiments. Statistical significance was calculated by 2-tailed Student’s t test (ns, not significant; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001).

    Techniques Used: Inhibition, Comparison, Flow Cytometry


    Figure Legend Snippet:

    Techniques Used: Recombinant, Membrane, Amplification, Mutagenesis, shRNA, Sequencing, Software

    z vad fmk  (Selleck Chemicals)


    Bioz Manufacturer Symbol Selleck Chemicals manufactures this product  
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    Selleck Chemicals z vad fmk
    scRNA-seq identified lethal levels of ROS in DTEPs after BET inhibition (A) Comparison of ROS pathway gene signature module scores on parental, DTP, DTEP-UN, and DTEP-TR. (B and C) Flow cytometry-based quantification of intracellular ROS level (mean fluorescent intensity) in on parental, DTP, DTEP-UN, and DTEP-TR in both PC9 (B) and A375 (C) cells. (Samples collected at 72 h, NEO2734: 0.25 μM). (D and E) Relative cell viability of DTEPs exposure to BET inhibitors with or without NAC, ferrostatin, and <t>Z-VAD-FMK</t> (NEO2734: 0.25 μM; NAC: 2.5 mM; ferrostatin: 2.5 μM; Z-VAD-FMK: 10 μM). Error bars in (B)–(E) represent mean ± SD, n = 3 independent experiments. Statistical significance was calculated by 2-tailed Student’s t test (ns, not significant; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001).
    Z Vad Fmk, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Targeting of vulnerabilities of drug-tolerant persisters identified through functional genetics delays tumor relapse"

    Article Title: Targeting of vulnerabilities of drug-tolerant persisters identified through functional genetics delays tumor relapse

    Journal: Cell Reports Medicine

    doi: 10.1016/j.xcrm.2024.101471

    scRNA-seq identified lethal levels of ROS in DTEPs after BET inhibition (A) Comparison of ROS pathway gene signature module scores on parental, DTP, DTEP-UN, and DTEP-TR. (B and C) Flow cytometry-based quantification of intracellular ROS level (mean fluorescent intensity) in on parental, DTP, DTEP-UN, and DTEP-TR in both PC9 (B) and A375 (C) cells. (Samples collected at 72 h, NEO2734: 0.25 μM). (D and E) Relative cell viability of DTEPs exposure to BET inhibitors with or without NAC, ferrostatin, and Z-VAD-FMK (NEO2734: 0.25 μM; NAC: 2.5 mM; ferrostatin: 2.5 μM; Z-VAD-FMK: 10 μM). Error bars in (B)–(E) represent mean ± SD, n = 3 independent experiments. Statistical significance was calculated by 2-tailed Student’s t test (ns, not significant; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001).
    Figure Legend Snippet: scRNA-seq identified lethal levels of ROS in DTEPs after BET inhibition (A) Comparison of ROS pathway gene signature module scores on parental, DTP, DTEP-UN, and DTEP-TR. (B and C) Flow cytometry-based quantification of intracellular ROS level (mean fluorescent intensity) in on parental, DTP, DTEP-UN, and DTEP-TR in both PC9 (B) and A375 (C) cells. (Samples collected at 72 h, NEO2734: 0.25 μM). (D and E) Relative cell viability of DTEPs exposure to BET inhibitors with or without NAC, ferrostatin, and Z-VAD-FMK (NEO2734: 0.25 μM; NAC: 2.5 mM; ferrostatin: 2.5 μM; Z-VAD-FMK: 10 μM). Error bars in (B)–(E) represent mean ± SD, n = 3 independent experiments. Statistical significance was calculated by 2-tailed Student’s t test (ns, not significant; ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001).

    Techniques Used: Inhibition, Comparison, Flow Cytometry


    Figure Legend Snippet:

    Techniques Used: Recombinant, Membrane, Amplification, Mutagenesis, shRNA, Sequencing, Software

    z vad fmk  (Selleck Chemicals)


    Bioz Manufacturer Symbol Selleck Chemicals manufactures this product  
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    Selleck Chemicals z vad fmk
    TRIM7 overexpression promoted the ferroptosis of GC cells. ( a ) TRIM7 overexpression reduced the cellular viability of GC cells. ( b ) Alive/dead cell viability assay showed that TRIM7 overexpression increased the cellular death. ( c ) Cellular death in TRIM7 overexpressing cells in the presence of different specific inhibitors such as Ferrostatin-1 (Fer-1), Liproxstatin-1 (Lip-1), <t>Z-VAD-FMK</t> (Z-VAD), Necrosulfonamide (NSA) and 3-Methyladenine (3-MA) at 48 h after transfection. ( d ) Alive/dead cell viability assay showed that cell death induced by overexpression of TRIM7 was reversed upon addition of the of the ferroptosis inhibitor Fer-1. ( e ) Lipid ROS in the TRIM7 overexpressing cells and control group after staining with BODIPY-C11. ( f , g ) Cellular MDA and 4-HNE expression in the TRIM7 overexpressing GC cells. *** P < 0.001; ns non-significant.
    Z Vad Fmk, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The E3 ligase TRIM7 suppresses the tumorigenesis of gastric cancer by targeting SLC7A11"

    Article Title: The E3 ligase TRIM7 suppresses the tumorigenesis of gastric cancer by targeting SLC7A11

    Journal: Scientific Reports

    doi: 10.1038/s41598-024-56746-3

    TRIM7 overexpression promoted the ferroptosis of GC cells. ( a ) TRIM7 overexpression reduced the cellular viability of GC cells. ( b ) Alive/dead cell viability assay showed that TRIM7 overexpression increased the cellular death. ( c ) Cellular death in TRIM7 overexpressing cells in the presence of different specific inhibitors such as Ferrostatin-1 (Fer-1), Liproxstatin-1 (Lip-1), Z-VAD-FMK (Z-VAD), Necrosulfonamide (NSA) and 3-Methyladenine (3-MA) at 48 h after transfection. ( d ) Alive/dead cell viability assay showed that cell death induced by overexpression of TRIM7 was reversed upon addition of the of the ferroptosis inhibitor Fer-1. ( e ) Lipid ROS in the TRIM7 overexpressing cells and control group after staining with BODIPY-C11. ( f , g ) Cellular MDA and 4-HNE expression in the TRIM7 overexpressing GC cells. *** P < 0.001; ns non-significant.
    Figure Legend Snippet: TRIM7 overexpression promoted the ferroptosis of GC cells. ( a ) TRIM7 overexpression reduced the cellular viability of GC cells. ( b ) Alive/dead cell viability assay showed that TRIM7 overexpression increased the cellular death. ( c ) Cellular death in TRIM7 overexpressing cells in the presence of different specific inhibitors such as Ferrostatin-1 (Fer-1), Liproxstatin-1 (Lip-1), Z-VAD-FMK (Z-VAD), Necrosulfonamide (NSA) and 3-Methyladenine (3-MA) at 48 h after transfection. ( d ) Alive/dead cell viability assay showed that cell death induced by overexpression of TRIM7 was reversed upon addition of the of the ferroptosis inhibitor Fer-1. ( e ) Lipid ROS in the TRIM7 overexpressing cells and control group after staining with BODIPY-C11. ( f , g ) Cellular MDA and 4-HNE expression in the TRIM7 overexpressing GC cells. *** P < 0.001; ns non-significant.

    Techniques Used: Over Expression, Viability Assay, Transfection, Staining, Expressing

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    Selleck Chemicals z vad fmk s7023
    Z Vad Fmk S7023, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Selleck Chemicals z vad fmk
    Compound heterozygous gain-of-function mutations in RIPK1 causes systemic autoinflammatory diseases. a , Pedigrees of a family with two patients carrying mutations at RIPK1 K377 and R390. Patient 1 (P1) and patient 2 (P2) were represented with filled symbols. b, Timeline of recurrent fever episodes in P2 over 5 weeks. Red dots represented increased temperatures during fever episodes and grey boxes represent the normal temperatures between flares. c, Representative sonographic image showing lymphadenopathy of P2. The diameters of right cervical lymph nodes were marked with dotted yellow line. d, Representative macroscopic image of dermatological manifestations in P1 during acute phase reaction, showing skin rashes (arrows). e, Whole blood cell counts (WBC), C-reactive protein (CRP) and serum amyloid A protein (SAA) were measured serially after the first evaluation of P1 and prednisone treatment (cyan shading) started at the age between 0-5. Horizontal dotted lines indicate age-specific high values for CRP, SAA or high and low values for WBC count. Red dots donate the abnormal values. f, C-reactive protein (CRP), serum ADA enzymatic activity and IL-6, TNF levels of unaffected controls (C, n=13), P1 during prednisone treatment (4 samples taken during flare and 5 samples taken during non-flare), P2 (4 samples taken during flare and 6 samples taken during non-flare), unaffected parents carrying heterozygous K377E mutation (K377E/WT, 1 sample from I-2 and 2 samples from II-2) or R390G mutation (R390G/WT, 3 samples from II-3). g, Sanger sequencing chromatograms of RIPK1 showed heterozygous base substitutions at p. K377E and p. R390G in whole blood genomic DNA from P1 and P2. h, WebLogo demonstrating conservation of human RIPK1 K377 and R390 across 42 vertebrate species. i-l, Cell death of SV40-immortalized human dermal fibroblasts (SV40-HDFs) isolated from three unaffected controls (C1-C3) and P2 were monitored by continuous imaging of SYTOX™ Orange staining. The SV40-HDFs were treated with T, S, Z as indicated ( i ). SV40-HDFs was treated with TZ with or without Nec-1s (10 μM) for 12 hours and immunoblotted with indicated antibodies ( j ). The TNFR signaling complex (TNF-RSC) were immunoprecipitated with Flag-TNF for 5 or 15 min and immunoblotted with indicated antibodies ( k ). NF-κB and MAPK activation of SV40-HDFs treated with TNF for 5, 15 or 30 min immunoblotted with indicated antibodies ( l ). m-p, RIPK1 -/- HT-29 cells were lentivirally complemented with HA-RIPK1 wild-type (WT), K377E, R390G and treated with T, S, Z as indicated. Cell viability was measured by CellTiter-Glo luminescent cell viability assay ( m ). The necroptosis markers ( n ), TNF-RSC ( o ) and the NF-κB and MAPK activation ( p ) were analyzed in the same way. T denotes 20 ng ml - TNF; S denotes 250 nM SM-164; Z denotes 25 μM <t>z-VAD-fmk.</t> Graphs show mean±SD. Statistical significance was determined by one-way ANOVA ( m ), **P < 0.01, ***P < 0.001.
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    Selleck Chemicals z vad fmk z vad
    Juglone induces cell death phenotypic changes in GBM cells. A LN229 and T98G cells were pretreated with inhibitors such as Fer-1 (4 μM), Lip-1 (0.1 μM), Nec-1 (10 μM), <t>Z-VAD</t> (25 μM), CQ-1(10 μM) or Mdivi(20 μM) for 1 h and then co-treated with juglone for 24 h. The cell viability was determined through a CCK-8 assay. B , C LN229 and T98G cells were treated with different concentrations of juglone for 24 h. The ROS levels of each group of cells were detected and quantitatively analyzed through flow cytometry. D , E The levels of GSH and MDA in LN229 and T98G cells were measured under the effect of different concentrations of juglone. F , G After 24 h of treatment with different concentrations of juglone, the 4HNE level in LN229 and T98G cells was detected and quantitatively analyzed through immunofluorescence. Fer-1 ferrostatin-1, Lip-1 liproxstatin-1, Nec necrostatin-1, Z-VAD <t>Z-VAD-FMK,</t> CQ-1 chloroquine, Mdivi Mdivi-1, GSH glutathione, MDA malonaldehyde; *p < 0.05; **p < 0.01; ***p < 0.001; ns, no statistical significance
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    Compound heterozygous gain-of-function mutations in RIPK1 causes systemic autoinflammatory diseases. a , Pedigrees of a family with two patients carrying mutations at RIPK1 K377 and R390. Patient 1 (P1) and patient 2 (P2) were represented with filled symbols. b, Timeline of recurrent fever episodes in P2 over 5 weeks. Red dots represented increased temperatures during fever episodes and grey boxes represent the normal temperatures between flares. c, Representative sonographic image showing lymphadenopathy of P2. The diameters of right cervical lymph nodes were marked with dotted yellow line. d, Representative macroscopic image of dermatological manifestations in P1 during acute phase reaction, showing skin rashes (arrows). e, Whole blood cell counts (WBC), C-reactive protein (CRP) and serum amyloid A protein (SAA) were measured serially after the first evaluation of P1 and prednisone treatment (cyan shading) started at the age between 0-5. Horizontal dotted lines indicate age-specific high values for CRP, SAA or high and low values for WBC count. Red dots donate the abnormal values. f, C-reactive protein (CRP), serum ADA enzymatic activity and IL-6, TNF levels of unaffected controls (C, n=13), P1 during prednisone treatment (4 samples taken during flare and 5 samples taken during non-flare), P2 (4 samples taken during flare and 6 samples taken during non-flare), unaffected parents carrying heterozygous K377E mutation (K377E/WT, 1 sample from I-2 and 2 samples from II-2) or R390G mutation (R390G/WT, 3 samples from II-3). g, Sanger sequencing chromatograms of RIPK1 showed heterozygous base substitutions at p. K377E and p. R390G in whole blood genomic DNA from P1 and P2. h, WebLogo demonstrating conservation of human RIPK1 K377 and R390 across 42 vertebrate species. i-l, Cell death of SV40-immortalized human dermal fibroblasts (SV40-HDFs) isolated from three unaffected controls (C1-C3) and P2 were monitored by continuous imaging of SYTOX™ Orange staining. The SV40-HDFs were treated with T, S, Z as indicated ( i ). SV40-HDFs was treated with TZ with or without Nec-1s (10 μM) for 12 hours and immunoblotted with indicated antibodies ( j ). The TNFR signaling complex (TNF-RSC) were immunoprecipitated with Flag-TNF for 5 or 15 min and immunoblotted with indicated antibodies ( k ). NF-κB and MAPK activation of SV40-HDFs treated with TNF for 5, 15 or 30 min immunoblotted with indicated antibodies ( l ). m-p, RIPK1 -/- HT-29 cells were lentivirally complemented with HA-RIPK1 wild-type (WT), K377E, R390G and treated with T, S, Z as indicated. Cell viability was measured by CellTiter-Glo luminescent cell viability assay ( m ). The necroptosis markers ( n ), TNF-RSC ( o ) and the NF-κB and MAPK activation ( p ) were analyzed in the same way. T denotes 20 ng ml - TNF; S denotes 250 nM SM-164; Z denotes 25 μM z-VAD-fmk. Graphs show mean±SD. Statistical significance was determined by one-way ANOVA ( m ), **P < 0.01, ***P < 0.001.

    Journal: medRxiv

    Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

    doi: 10.1101/2024.03.28.24304774

    Figure Lengend Snippet: Compound heterozygous gain-of-function mutations in RIPK1 causes systemic autoinflammatory diseases. a , Pedigrees of a family with two patients carrying mutations at RIPK1 K377 and R390. Patient 1 (P1) and patient 2 (P2) were represented with filled symbols. b, Timeline of recurrent fever episodes in P2 over 5 weeks. Red dots represented increased temperatures during fever episodes and grey boxes represent the normal temperatures between flares. c, Representative sonographic image showing lymphadenopathy of P2. The diameters of right cervical lymph nodes were marked with dotted yellow line. d, Representative macroscopic image of dermatological manifestations in P1 during acute phase reaction, showing skin rashes (arrows). e, Whole blood cell counts (WBC), C-reactive protein (CRP) and serum amyloid A protein (SAA) were measured serially after the first evaluation of P1 and prednisone treatment (cyan shading) started at the age between 0-5. Horizontal dotted lines indicate age-specific high values for CRP, SAA or high and low values for WBC count. Red dots donate the abnormal values. f, C-reactive protein (CRP), serum ADA enzymatic activity and IL-6, TNF levels of unaffected controls (C, n=13), P1 during prednisone treatment (4 samples taken during flare and 5 samples taken during non-flare), P2 (4 samples taken during flare and 6 samples taken during non-flare), unaffected parents carrying heterozygous K377E mutation (K377E/WT, 1 sample from I-2 and 2 samples from II-2) or R390G mutation (R390G/WT, 3 samples from II-3). g, Sanger sequencing chromatograms of RIPK1 showed heterozygous base substitutions at p. K377E and p. R390G in whole blood genomic DNA from P1 and P2. h, WebLogo demonstrating conservation of human RIPK1 K377 and R390 across 42 vertebrate species. i-l, Cell death of SV40-immortalized human dermal fibroblasts (SV40-HDFs) isolated from three unaffected controls (C1-C3) and P2 were monitored by continuous imaging of SYTOX™ Orange staining. The SV40-HDFs were treated with T, S, Z as indicated ( i ). SV40-HDFs was treated with TZ with or without Nec-1s (10 μM) for 12 hours and immunoblotted with indicated antibodies ( j ). The TNFR signaling complex (TNF-RSC) were immunoprecipitated with Flag-TNF for 5 or 15 min and immunoblotted with indicated antibodies ( k ). NF-κB and MAPK activation of SV40-HDFs treated with TNF for 5, 15 or 30 min immunoblotted with indicated antibodies ( l ). m-p, RIPK1 -/- HT-29 cells were lentivirally complemented with HA-RIPK1 wild-type (WT), K377E, R390G and treated with T, S, Z as indicated. Cell viability was measured by CellTiter-Glo luminescent cell viability assay ( m ). The necroptosis markers ( n ), TNF-RSC ( o ) and the NF-κB and MAPK activation ( p ) were analyzed in the same way. T denotes 20 ng ml - TNF; S denotes 250 nM SM-164; Z denotes 25 μM z-VAD-fmk. Graphs show mean±SD. Statistical significance was determined by one-way ANOVA ( m ), **P < 0.01, ***P < 0.001.

    Article Snippet: Recombinant human TNF (Peprotech, 300-01A) was used to stimulate HT-29 cells (20 ng ml - ), SV40- HDFs (20 ng ml - ) and human primary monocytes (50 ng ml - ) for the indicated times. z-VAD-fmk (Selleck, S7023, 25 μM), SM-164 (Selleck, S7089, 250 nM) and Nec-1s (Selleck, S8641, 10 μM) were used to treat HT-29 cells, human primary T cells and monocytes.

    Techniques: Activity Assay, Mutagenesis, Sequencing, Isolation, Imaging, Staining, Immunoprecipitation, Activation Assay, Cell Viability Assay

    RIPK1 mutations sensitize cells to TNF-induced cell death. a, SV40-HDFs isolated from three unaffected controls (C1-C3) and P2 were treated with T with or without S, Z and N as indicated. Cell death were monitored by imaging of SYTOX™ Orange staining. b-d, CellTiter-Glo luminescent cell viability detection of RIPK1 -/- HT-29 cells complemented with WT or mutated RIPK1 K377R, K377E, R390G, K377E/R390G under stimulation of TZ, TS, TSZ with or without N as indicated (b-c) . Expression levels of WT or mutated RIPK1 were determined with immunoblot (d) . e, mRNA levels of TNF , CXCL8 in complemented HT-29 cells treated with TNF for 6 hours were determined with qRT-PCR. f-g, HEK293T cells were transfected with plasmids encoding full length/intermediate domain of RIPK1 together with MIB2 or Ub as indicated. ID domain of RIPK1 or MIB2 were immunoprecipitated with anti-Flag dynabeads and immunoblotted with indicated antibodies. T denotes 20 ng ml - TNF; S denotes 250 nM SM-164; Z denotes 25 μM z-VAD-fmk; N denotes 10 μM Nec-1s. Graphs show mean±SD.

    Journal: medRxiv

    Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

    doi: 10.1101/2024.03.28.24304774

    Figure Lengend Snippet: RIPK1 mutations sensitize cells to TNF-induced cell death. a, SV40-HDFs isolated from three unaffected controls (C1-C3) and P2 were treated with T with or without S, Z and N as indicated. Cell death were monitored by imaging of SYTOX™ Orange staining. b-d, CellTiter-Glo luminescent cell viability detection of RIPK1 -/- HT-29 cells complemented with WT or mutated RIPK1 K377R, K377E, R390G, K377E/R390G under stimulation of TZ, TS, TSZ with or without N as indicated (b-c) . Expression levels of WT or mutated RIPK1 were determined with immunoblot (d) . e, mRNA levels of TNF , CXCL8 in complemented HT-29 cells treated with TNF for 6 hours were determined with qRT-PCR. f-g, HEK293T cells were transfected with plasmids encoding full length/intermediate domain of RIPK1 together with MIB2 or Ub as indicated. ID domain of RIPK1 or MIB2 were immunoprecipitated with anti-Flag dynabeads and immunoblotted with indicated antibodies. T denotes 20 ng ml - TNF; S denotes 250 nM SM-164; Z denotes 25 μM z-VAD-fmk; N denotes 10 μM Nec-1s. Graphs show mean±SD.

    Article Snippet: Recombinant human TNF (Peprotech, 300-01A) was used to stimulate HT-29 cells (20 ng ml - ), SV40- HDFs (20 ng ml - ) and human primary monocytes (50 ng ml - ) for the indicated times. z-VAD-fmk (Selleck, S7023, 25 μM), SM-164 (Selleck, S7089, 250 nM) and Nec-1s (Selleck, S8641, 10 μM) were used to treat HT-29 cells, human primary T cells and monocytes.

    Techniques: Isolation, Imaging, Staining, Expressing, Western Blot, Quantitative RT-PCR, Transfection, Immunoprecipitation

    Strong activation of inflammatory signaling in myeloid cells carrying RIPK1 mutations. a , Integrated uniform manifold approximation and projection (UMAP) visualization of peripheral blood mononuclear cells (PBMCs) from three unaffected controls (C1, n=13925 cells; C2, n=11117 cells; C3, n=12573 cells) and two patients (P1, n=10918 cells; P2, n=8516 cells), marker-based annotation of 15 cell subtypes were colored by cluster identity. LDG, low-density granulocyte; pDC, plasmacytoid dendritic cell; Mo-DC, monocyte-derived dendritic cell; NK, natural killer cell; NKT, natural killer T cell; Eryth, erythrocyte; MK, megakaryocyte. b, Visualization of expressions of IL1B and CXCL8 (colored single cells) projecting PBMCs from unaffected controls (C1-C3) and two patients (P1 and P2) on UMAP plots. c, Boxplots of the NF-κB expression score (top panel) and IFN response score (bottom panel) of cell subtypes. d, Heatmap of representative genes downstream of NF-κB and type Ι IFN pathways in monocyte compartments. Gene names were listed in Extended Data Fig.4d. e, Dot plot showing the biological processes identified with un-regulated gene terms (FDR <0.05) in patients’ monocytes compared to that of unaffected controls using DAVID. The size of the circle indicates the gene count enriched in the pathway and the color represents pathway enrichment significance. f, Intracellular cytokine analysis of IL-1β, IL-6 and IFNγ in monocytes isolated from three unaffected (C1- C3) controls and P1. g, qRT-PCR analysis of IL1B, IL6, CXCL8 and TNF mRNA levels in primary monocytes isolated from unaffected controls (n=8-10) and two patients (n=4 from P1 and P2) with or without 10 ng ml - TNF for 6 hours. h, CellTiter-Glo luminescent cell viability detection of primary monocytes isolated from unaffected controls (n=12) and two patients (n=3 from P1 and P2) stimulated with T, S, Z for indicated times. i, Statistics of dead M1 macrophages stimulated with TS or TSZ for 24 hours by imaging of propidium iodide (PI). M1 macrophages were differentiated from primary monocytes of unaffected controls (n=6) and two patients (n=4 from P1 and P2). Representative images were presented in Extended Data Fig.3e. j, Volcano plot displaying the differentially expressed genes in monocyte compartments between three unaffected controls (C1-C3) and two patients (P1 and P2). Representative suppressive genes of cell death were labeled with red dots and gene names. k, Clusters in the monocyte compartments showing the expression levels of TNFAIP3 and BCL2A1 in three unaffected controls (C1-C3) and two patients (P1 and P2). l, Schematic diagram showing serum cytokine microarray analysis of continuous P2’s serum samples before (PS1 and PS2) or after (PS3, flare; PS4 and PS5, non-flare) the onset of recurrent fever. m, PCA analysis of sample variance determined by semi-quantitative 174 cytokines antibody array on serum samples of C1-C3 from three unaffected controls and PS1-PS5 from P2. n, The integrated relative MFI plots of P2’s serum cytokine levels. 28 cytokines peaked in PS3 were enrich in group 1, 24 cytokines higher than control were enriched in group 2. o, Boxplots exhibiting mRNA expression scores of cytokines of group 1 (left panel) and group 2 (right panel) in indicated cell compartments. Group 1 and group 2 cytokine panels were clustered with the serum cytokine microarray analysis in Extended Data Fig.4. T denotes 10 ng ml - TNF; S denotes 250 nM SM164; Z denotes 25 μM z-VAD-fmk. All histogram graphs show mean±SD. Statistical significance was determined by two-tailed unpaired t-test ( c, g, i, l ) or one-way ANOVA ( h ), ns, P > 0.05, * P <0.05, ** P < 0.01, *** P < 0.001.

    Journal: medRxiv

    Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

    doi: 10.1101/2024.03.28.24304774

    Figure Lengend Snippet: Strong activation of inflammatory signaling in myeloid cells carrying RIPK1 mutations. a , Integrated uniform manifold approximation and projection (UMAP) visualization of peripheral blood mononuclear cells (PBMCs) from three unaffected controls (C1, n=13925 cells; C2, n=11117 cells; C3, n=12573 cells) and two patients (P1, n=10918 cells; P2, n=8516 cells), marker-based annotation of 15 cell subtypes were colored by cluster identity. LDG, low-density granulocyte; pDC, plasmacytoid dendritic cell; Mo-DC, monocyte-derived dendritic cell; NK, natural killer cell; NKT, natural killer T cell; Eryth, erythrocyte; MK, megakaryocyte. b, Visualization of expressions of IL1B and CXCL8 (colored single cells) projecting PBMCs from unaffected controls (C1-C3) and two patients (P1 and P2) on UMAP plots. c, Boxplots of the NF-κB expression score (top panel) and IFN response score (bottom panel) of cell subtypes. d, Heatmap of representative genes downstream of NF-κB and type Ι IFN pathways in monocyte compartments. Gene names were listed in Extended Data Fig.4d. e, Dot plot showing the biological processes identified with un-regulated gene terms (FDR <0.05) in patients’ monocytes compared to that of unaffected controls using DAVID. The size of the circle indicates the gene count enriched in the pathway and the color represents pathway enrichment significance. f, Intracellular cytokine analysis of IL-1β, IL-6 and IFNγ in monocytes isolated from three unaffected (C1- C3) controls and P1. g, qRT-PCR analysis of IL1B, IL6, CXCL8 and TNF mRNA levels in primary monocytes isolated from unaffected controls (n=8-10) and two patients (n=4 from P1 and P2) with or without 10 ng ml - TNF for 6 hours. h, CellTiter-Glo luminescent cell viability detection of primary monocytes isolated from unaffected controls (n=12) and two patients (n=3 from P1 and P2) stimulated with T, S, Z for indicated times. i, Statistics of dead M1 macrophages stimulated with TS or TSZ for 24 hours by imaging of propidium iodide (PI). M1 macrophages were differentiated from primary monocytes of unaffected controls (n=6) and two patients (n=4 from P1 and P2). Representative images were presented in Extended Data Fig.3e. j, Volcano plot displaying the differentially expressed genes in monocyte compartments between three unaffected controls (C1-C3) and two patients (P1 and P2). Representative suppressive genes of cell death were labeled with red dots and gene names. k, Clusters in the monocyte compartments showing the expression levels of TNFAIP3 and BCL2A1 in three unaffected controls (C1-C3) and two patients (P1 and P2). l, Schematic diagram showing serum cytokine microarray analysis of continuous P2’s serum samples before (PS1 and PS2) or after (PS3, flare; PS4 and PS5, non-flare) the onset of recurrent fever. m, PCA analysis of sample variance determined by semi-quantitative 174 cytokines antibody array on serum samples of C1-C3 from three unaffected controls and PS1-PS5 from P2. n, The integrated relative MFI plots of P2’s serum cytokine levels. 28 cytokines peaked in PS3 were enrich in group 1, 24 cytokines higher than control were enriched in group 2. o, Boxplots exhibiting mRNA expression scores of cytokines of group 1 (left panel) and group 2 (right panel) in indicated cell compartments. Group 1 and group 2 cytokine panels were clustered with the serum cytokine microarray analysis in Extended Data Fig.4. T denotes 10 ng ml - TNF; S denotes 250 nM SM164; Z denotes 25 μM z-VAD-fmk. All histogram graphs show mean±SD. Statistical significance was determined by two-tailed unpaired t-test ( c, g, i, l ) or one-way ANOVA ( h ), ns, P > 0.05, * P <0.05, ** P < 0.01, *** P < 0.001.

    Article Snippet: Recombinant human TNF (Peprotech, 300-01A) was used to stimulate HT-29 cells (20 ng ml - ), SV40- HDFs (20 ng ml - ) and human primary monocytes (50 ng ml - ) for the indicated times. z-VAD-fmk (Selleck, S7023, 25 μM), SM-164 (Selleck, S7089, 250 nM) and Nec-1s (Selleck, S8641, 10 μM) were used to treat HT-29 cells, human primary T cells and monocytes.

    Techniques: Activation Assay, Marker, Derivative Assay, Expressing, Isolation, Quantitative RT-PCR, Imaging, Labeling, Microarray, Ab Array, Two Tailed Test

    Increased cell death and CD4/CD8 ratio in T cells caused by RIPK1 activation. a , Average expression values (arcsinh transformed) of cl. Casp-3 across identified peripheral immune cell lineages in PBMCs freshly isolated from four unaffected controls (C) and two patients (P, 1 sample from P1 and 2 samples from P2) by mass cytometry by time-of-flight (CyTOF) analysis. b-e, Representative contour plots of cl. Casp-3 ( b-c ) and histograms of total RIPK1, pRIPK1(S166) ( d-e ) levels in CD4 + T and CD8 + T cells in freshly isolated PBMCs from unaffected controls (n=10-12) and patients (n=4 for P1 and P2) (left panel). Summary histograms of cl. Casp-3 and pRIPK1(S166) (right panel) were shown. f, Immunoblotting analysis of primary CD3 + T cells isolated from two unaffected controls (C1 and C2) and P1 treated with T and S with or without Z, N for 6 hours. Antibodies as indicated were immunoblotted to show the activation of apoptosis or necroptosis. g, CellTiter-Glo luminescent cell viability detection of primary CD3 + T cells isolated from three unaffected controls (C1-C3), two patients (P1 and P2) and their parents treated with TS or TSZ for indicated times, n=4. h, Representative histogram and statistical analysis of surface CD69 expression on CD3 + T cells from unaffected controls (n=7) and two patients (n=3 for P1 and P2). i, Serum IFNγ, sCD25, sCD95 and CD95L of unaffected controls (C, n=10), two patients (P1, n=5-7; P2, n=4-8) and their parents (n=6-7) as well as two CRIA syndrome patients (n=6) determined by ELISA. j, CD4/CD8 ratio in T cells, percentages and absolute counts of CD3 + T, CD4 + T, CD8 + T cells in whole blood sample of P1 (red dots) and P2 (blue dots) measured using immunophenotyping. k, In vitro expansion of primary T cells isolated from unaffected controls (n=7) and patients (n=3 for P1 and P2) as indicated. Cell numbers were counted every two days post anti-CD3/CD28 dynabeads stimulation. l, Proliferation of CD4 + T and CD8 + T cells isolated from three unaffected controls (C1-C3) and P1 were analyzed using CFSE staining upon stimulation with anti-CD3/CD28 dynabeads for 96 hours. m, Representative histograms of surface CD95 expression on CD3 + T cells analyzed with flow cytometry after culturing in vitro for 24 hours. n, Representative contour plots depicting the percentages of CD4 + T, CD8 + T, double negative T (DNT) and double positive T (DPT) in CD3 + T cells gated from PBMCs of P2 culturing in vitro for 24 hours. Histograms displaying cl. Casp-3 and RIPK1 expression levels in CD8 hi T and CD8 lo T cells. o, Representative contour plots and summary histograms showing percentages of CD4 + T, CD8 + T and CD8 hi T cells and CD4/CD8 ratio in CD3 + T cells isolated from P2 cultured for indicated times. p-q, Representative contour plots showing percentages of CD4 + T, CD8 + T in CD3 + T cells of P2 with (TurboGFP + ) or without (TurboGFP - ) restoration using CRISPR/Cas9-mediated in situ knock-in of WT RIPK1-P2A-TurboGFP under stimulation of T with or without S, Z for 6 hours ( p ). CD4/CD8 ratio in CD3 + T cells of unaffected controls (n=7) and two patients (n=3 for P1 and P2) were quantified ( q ). r-s, Representative contour plots ( r ) and statistical analysis ( s ) showing percentages of CD4 + T, CD8 + T, DNT and DPT cell in CD3 + T cells isolated from unaffected control (n=7) and two patients (n=9 for P1 and P2) under the stimulation of anti-CD3 antibody (5 μg ml - ) for 72 hours. t, Statistical analysis of fold change of CD4 /CD8 ratio in CD3 + T cells isolated from two patients (n=4 for P1 and P2) under stimulation of anti-CD3 antibody (5 μg ml - ) with or without 250 nM Ponatinib or 10 μM Nec-1s for 24 hours. For representative contour plots, see Extended Data Fig. 6h. T denotes 10 ng ml - TNF; S denotes 250 nM SM164; Z denotes 25 μM z-VAD-fmk; N denotes 10 μM Nec-1s. All histogram graphs show mean±SD. Statistical significance was determined by one-way ANOVA ( g) or two-tailed unpaired t-test in the rest, * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

    Journal: medRxiv

    Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

    doi: 10.1101/2024.03.28.24304774

    Figure Lengend Snippet: Increased cell death and CD4/CD8 ratio in T cells caused by RIPK1 activation. a , Average expression values (arcsinh transformed) of cl. Casp-3 across identified peripheral immune cell lineages in PBMCs freshly isolated from four unaffected controls (C) and two patients (P, 1 sample from P1 and 2 samples from P2) by mass cytometry by time-of-flight (CyTOF) analysis. b-e, Representative contour plots of cl. Casp-3 ( b-c ) and histograms of total RIPK1, pRIPK1(S166) ( d-e ) levels in CD4 + T and CD8 + T cells in freshly isolated PBMCs from unaffected controls (n=10-12) and patients (n=4 for P1 and P2) (left panel). Summary histograms of cl. Casp-3 and pRIPK1(S166) (right panel) were shown. f, Immunoblotting analysis of primary CD3 + T cells isolated from two unaffected controls (C1 and C2) and P1 treated with T and S with or without Z, N for 6 hours. Antibodies as indicated were immunoblotted to show the activation of apoptosis or necroptosis. g, CellTiter-Glo luminescent cell viability detection of primary CD3 + T cells isolated from three unaffected controls (C1-C3), two patients (P1 and P2) and their parents treated with TS or TSZ for indicated times, n=4. h, Representative histogram and statistical analysis of surface CD69 expression on CD3 + T cells from unaffected controls (n=7) and two patients (n=3 for P1 and P2). i, Serum IFNγ, sCD25, sCD95 and CD95L of unaffected controls (C, n=10), two patients (P1, n=5-7; P2, n=4-8) and their parents (n=6-7) as well as two CRIA syndrome patients (n=6) determined by ELISA. j, CD4/CD8 ratio in T cells, percentages and absolute counts of CD3 + T, CD4 + T, CD8 + T cells in whole blood sample of P1 (red dots) and P2 (blue dots) measured using immunophenotyping. k, In vitro expansion of primary T cells isolated from unaffected controls (n=7) and patients (n=3 for P1 and P2) as indicated. Cell numbers were counted every two days post anti-CD3/CD28 dynabeads stimulation. l, Proliferation of CD4 + T and CD8 + T cells isolated from three unaffected controls (C1-C3) and P1 were analyzed using CFSE staining upon stimulation with anti-CD3/CD28 dynabeads for 96 hours. m, Representative histograms of surface CD95 expression on CD3 + T cells analyzed with flow cytometry after culturing in vitro for 24 hours. n, Representative contour plots depicting the percentages of CD4 + T, CD8 + T, double negative T (DNT) and double positive T (DPT) in CD3 + T cells gated from PBMCs of P2 culturing in vitro for 24 hours. Histograms displaying cl. Casp-3 and RIPK1 expression levels in CD8 hi T and CD8 lo T cells. o, Representative contour plots and summary histograms showing percentages of CD4 + T, CD8 + T and CD8 hi T cells and CD4/CD8 ratio in CD3 + T cells isolated from P2 cultured for indicated times. p-q, Representative contour plots showing percentages of CD4 + T, CD8 + T in CD3 + T cells of P2 with (TurboGFP + ) or without (TurboGFP - ) restoration using CRISPR/Cas9-mediated in situ knock-in of WT RIPK1-P2A-TurboGFP under stimulation of T with or without S, Z for 6 hours ( p ). CD4/CD8 ratio in CD3 + T cells of unaffected controls (n=7) and two patients (n=3 for P1 and P2) were quantified ( q ). r-s, Representative contour plots ( r ) and statistical analysis ( s ) showing percentages of CD4 + T, CD8 + T, DNT and DPT cell in CD3 + T cells isolated from unaffected control (n=7) and two patients (n=9 for P1 and P2) under the stimulation of anti-CD3 antibody (5 μg ml - ) for 72 hours. t, Statistical analysis of fold change of CD4 /CD8 ratio in CD3 + T cells isolated from two patients (n=4 for P1 and P2) under stimulation of anti-CD3 antibody (5 μg ml - ) with or without 250 nM Ponatinib or 10 μM Nec-1s for 24 hours. For representative contour plots, see Extended Data Fig. 6h. T denotes 10 ng ml - TNF; S denotes 250 nM SM164; Z denotes 25 μM z-VAD-fmk; N denotes 10 μM Nec-1s. All histogram graphs show mean±SD. Statistical significance was determined by one-way ANOVA ( g) or two-tailed unpaired t-test in the rest, * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

    Article Snippet: Recombinant human TNF (Peprotech, 300-01A) was used to stimulate HT-29 cells (20 ng ml - ), SV40- HDFs (20 ng ml - ) and human primary monocytes (50 ng ml - ) for the indicated times. z-VAD-fmk (Selleck, S7023, 25 μM), SM-164 (Selleck, S7089, 250 nM) and Nec-1s (Selleck, S8641, 10 μM) were used to treat HT-29 cells, human primary T cells and monocytes.

    Techniques: Activation Assay, Expressing, Transformation Assay, Isolation, Mass Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, In Vitro, Staining, Flow Cytometry, Cell Culture, CRISPR, In Situ, Knock-In, Two Tailed Test

    RIPK1-mediated cell death leads to CD8 decrease in T cells. a, Percentages and absolute counts of total lymphocytes, γδ T cells, NKT cells and B cells from whole blood of P1 (red dots) and P2 (blue dots) measured by immunophenotyping. b, Representative contour plots showing percentages of CD8 hi T, CD8 lo T, CD4 + T, DNT and DPT cells in CD3 + T cells isolated from unaffected control and P1 under the stimulation of TS, TSZ and cytokines (combination of IL-1β (10 ng ml - ), IL-6 (100 ng ml - ), IFNα (100 ng ml - ) and IL-8 (100 ng ml - )) as indicated for 6 hours. c- d, Representative contour plots ( c ) and summary histogram ( d ) showing percentages of CD8 hi T, CD8 lo T, CD4 + T, DNT and DPT cells in CD3 + T cells isolated from P2 under the stimulation of TS, TSZ, TSN and TSZN as indicated for 6 hours. e, Schematic of CRISPR/Cas9-mediated in situ knock-in of WT RIPK1 into patients’ RIPK1 locus. f, Representative contour plots of Turbo-GFP + cells percentage indicating WT RIPK1 knockin efficiency. g, Statistical analysis of CD8 + T cell proportions in CD3 + T cells from unaffected controls (n=3-6) and two patients (n=3 for P1 and P2) under the stimulation of TS, TSZ as well as RSL3 (1 μM) and STS (100 nM) for indicated times. h, Representative contour plots showing percentages of CD8 + T and CD4 + T cells in CD3 + T cells isolated from P2 under the stimulation of anti-CD3 antibody (5 μg/ml) with or without Ponatinib (250 nM) and Nec-1s (10 μM) for 24 hours. T denotes 20 ng ml - TNF; S denotes 250 nM SM-164; Z denotes 25 μM z-VAD-fmk; N denotes 10 μM Nec-1s. Graphs show mean±SD.

    Journal: medRxiv

    Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

    doi: 10.1101/2024.03.28.24304774

    Figure Lengend Snippet: RIPK1-mediated cell death leads to CD8 decrease in T cells. a, Percentages and absolute counts of total lymphocytes, γδ T cells, NKT cells and B cells from whole blood of P1 (red dots) and P2 (blue dots) measured by immunophenotyping. b, Representative contour plots showing percentages of CD8 hi T, CD8 lo T, CD4 + T, DNT and DPT cells in CD3 + T cells isolated from unaffected control and P1 under the stimulation of TS, TSZ and cytokines (combination of IL-1β (10 ng ml - ), IL-6 (100 ng ml - ), IFNα (100 ng ml - ) and IL-8 (100 ng ml - )) as indicated for 6 hours. c- d, Representative contour plots ( c ) and summary histogram ( d ) showing percentages of CD8 hi T, CD8 lo T, CD4 + T, DNT and DPT cells in CD3 + T cells isolated from P2 under the stimulation of TS, TSZ, TSN and TSZN as indicated for 6 hours. e, Schematic of CRISPR/Cas9-mediated in situ knock-in of WT RIPK1 into patients’ RIPK1 locus. f, Representative contour plots of Turbo-GFP + cells percentage indicating WT RIPK1 knockin efficiency. g, Statistical analysis of CD8 + T cell proportions in CD3 + T cells from unaffected controls (n=3-6) and two patients (n=3 for P1 and P2) under the stimulation of TS, TSZ as well as RSL3 (1 μM) and STS (100 nM) for indicated times. h, Representative contour plots showing percentages of CD8 + T and CD4 + T cells in CD3 + T cells isolated from P2 under the stimulation of anti-CD3 antibody (5 μg/ml) with or without Ponatinib (250 nM) and Nec-1s (10 μM) for 24 hours. T denotes 20 ng ml - TNF; S denotes 250 nM SM-164; Z denotes 25 μM z-VAD-fmk; N denotes 10 μM Nec-1s. Graphs show mean±SD.

    Article Snippet: Recombinant human TNF (Peprotech, 300-01A) was used to stimulate HT-29 cells (20 ng ml - ), SV40- HDFs (20 ng ml - ) and human primary monocytes (50 ng ml - ) for the indicated times. z-VAD-fmk (Selleck, S7023, 25 μM), SM-164 (Selleck, S7089, 250 nM) and Nec-1s (Selleck, S8641, 10 μM) were used to treat HT-29 cells, human primary T cells and monocytes.

    Techniques: Isolation, CRISPR, In Situ, Knock-In

    RIPK1 activation causes inflammation through T-Mono axis. a , Schematic diagram of T cell-MDM co-culture system. b-c, qRT-PCR analysis of TNF, IL1B and IL6 mRNA levels in GM-CSF-activated monocytes derived macrophages (MDMs) co-cultured with or without T cells for 6 hours. GM-CSF-activated MDMs generated from unaffected control 1 (C1) (left panel) or unaffected control 3 (C3) (right panel) ( b ) or P1 ( c ) were co-cultured with donor-matched T cells as well as T cells from other unaffected controls (C2 and C4) or patients as indicated. d, Dot plot of IFNG, TNF in cell subtypes found in scRNA-seq of PBMCs from three unaffected controls and two patients. The size of the circle indicates the percentage of cells positive for gene expression and the color represents the average expression level. e, Representative histograms and statistical analysis of TNF and IFNγ expression in CD8 + T cells in PBMCs isolated from unaffected controls (n=6) and patients (n=3 for P1 and P2) treated with 10 ng ml - TNF (T) with or without 250 nM SM-164 (S), 25 μM z-VAD-fmk (Z) for 24 hours. f, T cells isolated from P2 were co-cultured with MDMs generated from unaffected control in the presence of 10 μg ml - adalimumab, 10 μg ml - emapalumab alone or in combination for 6 hours. TNF, IL1B and IL6 mRNA levels of MDMs were analyzed by qRT-PCR. g, Experimental timeline for generation of HSCs-transplanted mice with conditional overexpression of human WT or mutated RIPK1 K377E, R390G or D324V in CD8α + T cell- or myeloid cell-specific lineage. h-i, ELISA measurement of serum Tnf and Il6 levels in mice specifically overexpressing WT or mutated hRIPK1 in CD8α + T cells ( h ) or myeloid cells ( i ). j-m, Pedigrees of two families with CRIA patients (CRIA-1 and CRIA-2) ( j ). Patients were represented with filled symbols. Summary histograms of CD4/CD8 ratio in CD3 + T cells of unaffected controls (n=12), CRIA-1 (n=7) and CRIA-2 (n=5) were shown ( k). Representative contour plots of cl. Casp-3 ( l ) and histograms of pRIPK1(S166) ( m ) protein levels in CD8 + T cells in freshly isolated PBMCs from CRIA patients. n-o, Whole blood cell counts (WBC), cell counts and proportions of neutrophil (NE#-NE%) and lymphocyte (LYMPH# -LYMPH%) ( n) , Serum C-reactive protein (CRP), TNF and IL-6 ( o ) of P1 (red dots) and P2 (blue dots) measured before and after tocilizumab or adalimumab treatments. The grey area represents the range of unaffected controls. Dots represent each sample and graphs show mean±SD. Statistical significance was determined by two-tailed unpaired t-test, * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

    Journal: medRxiv

    Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

    doi: 10.1101/2024.03.28.24304774

    Figure Lengend Snippet: RIPK1 activation causes inflammation through T-Mono axis. a , Schematic diagram of T cell-MDM co-culture system. b-c, qRT-PCR analysis of TNF, IL1B and IL6 mRNA levels in GM-CSF-activated monocytes derived macrophages (MDMs) co-cultured with or without T cells for 6 hours. GM-CSF-activated MDMs generated from unaffected control 1 (C1) (left panel) or unaffected control 3 (C3) (right panel) ( b ) or P1 ( c ) were co-cultured with donor-matched T cells as well as T cells from other unaffected controls (C2 and C4) or patients as indicated. d, Dot plot of IFNG, TNF in cell subtypes found in scRNA-seq of PBMCs from three unaffected controls and two patients. The size of the circle indicates the percentage of cells positive for gene expression and the color represents the average expression level. e, Representative histograms and statistical analysis of TNF and IFNγ expression in CD8 + T cells in PBMCs isolated from unaffected controls (n=6) and patients (n=3 for P1 and P2) treated with 10 ng ml - TNF (T) with or without 250 nM SM-164 (S), 25 μM z-VAD-fmk (Z) for 24 hours. f, T cells isolated from P2 were co-cultured with MDMs generated from unaffected control in the presence of 10 μg ml - adalimumab, 10 μg ml - emapalumab alone or in combination for 6 hours. TNF, IL1B and IL6 mRNA levels of MDMs were analyzed by qRT-PCR. g, Experimental timeline for generation of HSCs-transplanted mice with conditional overexpression of human WT or mutated RIPK1 K377E, R390G or D324V in CD8α + T cell- or myeloid cell-specific lineage. h-i, ELISA measurement of serum Tnf and Il6 levels in mice specifically overexpressing WT or mutated hRIPK1 in CD8α + T cells ( h ) or myeloid cells ( i ). j-m, Pedigrees of two families with CRIA patients (CRIA-1 and CRIA-2) ( j ). Patients were represented with filled symbols. Summary histograms of CD4/CD8 ratio in CD3 + T cells of unaffected controls (n=12), CRIA-1 (n=7) and CRIA-2 (n=5) were shown ( k). Representative contour plots of cl. Casp-3 ( l ) and histograms of pRIPK1(S166) ( m ) protein levels in CD8 + T cells in freshly isolated PBMCs from CRIA patients. n-o, Whole blood cell counts (WBC), cell counts and proportions of neutrophil (NE#-NE%) and lymphocyte (LYMPH# -LYMPH%) ( n) , Serum C-reactive protein (CRP), TNF and IL-6 ( o ) of P1 (red dots) and P2 (blue dots) measured before and after tocilizumab or adalimumab treatments. The grey area represents the range of unaffected controls. Dots represent each sample and graphs show mean±SD. Statistical significance was determined by two-tailed unpaired t-test, * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

    Article Snippet: Recombinant human TNF (Peprotech, 300-01A) was used to stimulate HT-29 cells (20 ng ml - ), SV40- HDFs (20 ng ml - ) and human primary monocytes (50 ng ml - ) for the indicated times. z-VAD-fmk (Selleck, S7023, 25 μM), SM-164 (Selleck, S7089, 250 nM) and Nec-1s (Selleck, S8641, 10 μM) were used to treat HT-29 cells, human primary T cells and monocytes.

    Techniques: Activation Assay, Co-Culture Assay, Quantitative RT-PCR, Derivative Assay, Cell Culture, Generated, Expressing, Isolation, Over Expression, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Juglone induces cell death phenotypic changes in GBM cells. A LN229 and T98G cells were pretreated with inhibitors such as Fer-1 (4 μM), Lip-1 (0.1 μM), Nec-1 (10 μM), Z-VAD (25 μM), CQ-1(10 μM) or Mdivi(20 μM) for 1 h and then co-treated with juglone for 24 h. The cell viability was determined through a CCK-8 assay. B , C LN229 and T98G cells were treated with different concentrations of juglone for 24 h. The ROS levels of each group of cells were detected and quantitatively analyzed through flow cytometry. D , E The levels of GSH and MDA in LN229 and T98G cells were measured under the effect of different concentrations of juglone. F , G After 24 h of treatment with different concentrations of juglone, the 4HNE level in LN229 and T98G cells was detected and quantitatively analyzed through immunofluorescence. Fer-1 ferrostatin-1, Lip-1 liproxstatin-1, Nec necrostatin-1, Z-VAD Z-VAD-FMK, CQ-1 chloroquine, Mdivi Mdivi-1, GSH glutathione, MDA malonaldehyde; *p < 0.05; **p < 0.01; ***p < 0.001; ns, no statistical significance

    Journal: Chinese Medicine

    Article Title: Juglone induces ferroptosis in glioblastoma cells by inhibiting the Nrf2-GPX4 axis through the phosphorylation of p38MAPK

    doi: 10.1186/s13020-024-00920-2

    Figure Lengend Snippet: Juglone induces cell death phenotypic changes in GBM cells. A LN229 and T98G cells were pretreated with inhibitors such as Fer-1 (4 μM), Lip-1 (0.1 μM), Nec-1 (10 μM), Z-VAD (25 μM), CQ-1(10 μM) or Mdivi(20 μM) for 1 h and then co-treated with juglone for 24 h. The cell viability was determined through a CCK-8 assay. B , C LN229 and T98G cells were treated with different concentrations of juglone for 24 h. The ROS levels of each group of cells were detected and quantitatively analyzed through flow cytometry. D , E The levels of GSH and MDA in LN229 and T98G cells were measured under the effect of different concentrations of juglone. F , G After 24 h of treatment with different concentrations of juglone, the 4HNE level in LN229 and T98G cells was detected and quantitatively analyzed through immunofluorescence. Fer-1 ferrostatin-1, Lip-1 liproxstatin-1, Nec necrostatin-1, Z-VAD Z-VAD-FMK, CQ-1 chloroquine, Mdivi Mdivi-1, GSH glutathione, MDA malonaldehyde; *p < 0.05; **p < 0.01; ***p < 0.001; ns, no statistical significance

    Article Snippet: Juglone, Chloroquine (CQ-1), Ferrostatain-1 (Fer-1), Liproxstatin-1 (Lip-1), Mdivi-1 (Mdivi), Necrostatin-1 (Nec-1) and Z-VAD-FMK (Z-VAD) were purchased from Selleck.

    Techniques: CCK-8 Assay, Flow Cytometry, Immunofluorescence