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ripk1 inhibitor nec 1 (MedChemExpress)

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MedChemExpress ripk1 inhibitor nec 1
Ripk1 Inhibitor Nec 1, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 99/100, based on 778 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/ripk1+inhibitor+nec+1/Necrostatin-1/bio_rxiv__64898__2026__02__13__705677-46-50-68
Average 99 stars, based on 778 article reviews

ripk1 inhibitor nec 1 - by Bioz Stars, 2026-09

99/100 stars

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Recombinant:

Article Title: 5-Iodotubercidin inhibits Epithelial to Mesenchymal Transition by inhibiting IKK/NFκB-dependent gene expression
Article Snippet: Other chemicals and reagents used were: DAPI (#80145, Sisco Research Laboratories), ATP (#RM439, HiMedia), DiR (DiIC18) (#60017, Biotium Inc.). .. The source and working concentrations used for the stimulants and inhibitors were: recombinant human TGFβ (#100-21-10, Peprotech, 5 ng/mL) and recombinant human TNFα (#PHC3016, Gibco, 101ng/ml), 5-Iodotubercidin (#HY-15424, 0.1-10 μM), IKKα/β inhibitor BMS345541 Hydrochloride (#HY-10518, 5 μM) (IKKβ IC 50 = 0.3 μM, IKKα IC 50 = 4 μM) , RIPK1 inhibitor Nec-1 (#HY-15760, 50 μM), GSK481 (HY-100131, 5 μM), ABT-702 dihydrochloride (#HY-103161, 5-10 μM) were obtained from MedChemExpress. ..

Western Blot:

Article Title: Beneficial effects of aloperine on inflammation and oxidative stress by suppressing necroptosis in lipopolysaccharide-induced acute lung injury mouse model.
Article Snippet: Rationale: Alveolar epithelial cell death, inflammation, and oxidative stress are typical features of acute lung injury (ALI).. Aloperine (Alo), an alkaloid isolated from Sophora alopecuroides, has been reported to display various biological effects, such as anti-inflammatory, immunoregulatory, and anti-oxidant properties.. In this study, we investigated the effects and mechanisms of Alo in treating a lipopolysaccharide (LPS)-induced ALI in a

Phospho-proteomics:

Concentration Assay:

Article Title: The Bcr-Abl inhibitor GNF-7 inhibits necroptosis and ameliorates acute kidney injury by targeting RIPK1 and RIPK3 kinases.
Article Snippet: Necroptosis is a form of programmed, caspase-independent cell death that is involved in various pathologic disorders such as ischemia/reperfusion injury, acute kidney injury and inflammatory bowel diseases.. Identification of necroptosis inhibitors has great therapeutic potential for the treatment of necroptosis-associated diseases.. In this study, we identified that the Bcr-Abl inhibitor GNF-7 was a potent inhibitor of necroptosis.

Image Search Results

( A – C ) SUIT-020 were pretreated with BV6, zVAD-fmk, Nec-1s and GSK872 with the indicated concentrations for 1 h. Afterwards, the cells were treated with 500 ng/ml CD95L for 24 h. Cell viability was measured using the Cell Titer-Glo®-Luminescent Cell Viability Assay by Promega. The cell viability of untreated cells was taken as 100%. ( D , E ) Jurkat RIPK1 ko cells were pretreated for 1 h with 5 µM BV6, 50 µM zVAD-fmk and 10 µM Nec-1s. Afterwards, the cells were treated with 500 ng/ml CD95L for the indicated time intervals. Cells were analyzed via imaging flow cytometry. Populations were gated for viable (negative), Annexin-V-FITC positive and double-positive (Annexin-V-FITC and PI positive) cells. Representative pictures from imaging flow cytometry with viable (negative) Annexin-V-FITC positive and double-positive (Annexin-V-FITC and PI positive) cells are shown in the middle. Mean and standard deviation from three independent experiments are shown. Statistics were calculated with unpaired one-way ANOVA with Tukey post hoc test to compare two conditions. Significance values: **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05; ns not significant. P values from left to right for ( A ) P = 0.0030, P = 0.0281, P = 0.0198, P = 0.0403 ( B ) P = 0.0173, P = 0.0002, P < 0.0001 ( C ) P = 0.0033, P = 0.0006 ( D ) P > 0.9999 ( E ) p > 0.9999. ( F ) Total cellular lysates of MV4-11, HeLa-CD95 and SUIT-020 cells were tested for RIPK3 expression using western blot. HeLa-CD95 cells do not express RIPK3 and were used as a negative control. One representative western blot out of two is shown. Actin served as loading control. ( G ) SUIT-020 cells were with 500 ng/ml CD95L for indicated timepoints and DISC IP was analyzed by western blot. Actin served as loading control. BC beads-only control, IP immunoprecipitation, bf bright field, AVF Annexin-V-FITC, PI Propidium Iodide, * unspecific band, IgG H the heavy chain of antibody. .

Journal: The EMBO Journal

Article Title: Oligomerised RIPK1 is the main core component of the CD95 necrosome

doi: 10.1038/s44318-025-00433-0

Figure Lengend Snippet: ( A – C ) SUIT-020 were pretreated with BV6, zVAD-fmk, Nec-1s and GSK872 with the indicated concentrations for 1 h. Afterwards, the cells were treated with 500 ng/ml CD95L for 24 h. Cell viability was measured using the Cell Titer-Glo®-Luminescent Cell Viability Assay by Promega. The cell viability of untreated cells was taken as 100%. ( D , E ) Jurkat RIPK1 ko cells were pretreated for 1 h with 5 µM BV6, 50 µM zVAD-fmk and 10 µM Nec-1s. Afterwards, the cells were treated with 500 ng/ml CD95L for the indicated time intervals. Cells were analyzed via imaging flow cytometry. Populations were gated for viable (negative), Annexin-V-FITC positive and double-positive (Annexin-V-FITC and PI positive) cells. Representative pictures from imaging flow cytometry with viable (negative) Annexin-V-FITC positive and double-positive (Annexin-V-FITC and PI positive) cells are shown in the middle. Mean and standard deviation from three independent experiments are shown. Statistics were calculated with unpaired one-way ANOVA with Tukey post hoc test to compare two conditions. Significance values: **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05; ns not significant. P values from left to right for ( A ) P = 0.0030, P = 0.0281, P = 0.0198, P = 0.0403 ( B ) P = 0.0173, P = 0.0002, P < 0.0001 ( C ) P = 0.0033, P = 0.0006 ( D ) P > 0.9999 ( E ) p > 0.9999. ( F ) Total cellular lysates of MV4-11, HeLa-CD95 and SUIT-020 cells were tested for RIPK3 expression using western blot. HeLa-CD95 cells do not express RIPK3 and were used as a negative control. One representative western blot out of two is shown. Actin served as loading control. ( G ) SUIT-020 cells were with 500 ng/ml CD95L for indicated timepoints and DISC IP was analyzed by western blot. Actin served as loading control. BC beads-only control, IP immunoprecipitation, bf bright field, AVF Annexin-V-FITC, PI Propidium Iodide, * unspecific band, IgG H the heavy chain of antibody. .

Article Snippet: The RIPK1 inhibitor 7-Cl-O-Nec-1 (Nec-1s; 5.04297.0001) and the RIPK3 inhibitor GSK872 (5.30389) were obtained from Merck, Germany.

Techniques: Cell Viability Assay, Imaging, Flow Cytometry, Standard Deviation, Expressing, Western Blot, Negative Control, Control, Immunoprecipitation

( A – E ) HT29 cells ( A , B ), SUIT-020 cells ( C , D ), Jurkat 282 and Jurkat C8 ko cells ( E ) and Jurkat A3 and Jurkat RIPK1 ko cells ( F ) were pretreated for 1 h with 5 µM BV6, 50 µM zVAD-fmk and 10 µM Nec-1s and subsequently treated with 500 ng/ml CD95L for indicated timepoints ( B , C ), 2 h ( C ) or 5 h ( A , E , F ). pRIPK1- and RIPK1-IPs were carried out using anti-pRIPK1 and anti-RIPK1 antibodies, respectively, and analyzed by western blot. Actin was used as a loading control for the total cellular lysates (input). “Beads-only” pulldown (BC) was used as a negative control for IPs. One representative experiment out of three ( A , B , D – F ) or two ( C ) is shown. s.e.-short exposure, l.e. long exposure, IP immunoprecipitation, BC beads-only control pulldown, * unspecific band, IgG H the heavy chain of antibody. .

Journal: The EMBO Journal

Article Title: Oligomerised RIPK1 is the main core component of the CD95 necrosome

doi: 10.1038/s44318-025-00433-0

Figure Lengend Snippet: ( A – E ) HT29 cells ( A , B ), SUIT-020 cells ( C , D ), Jurkat 282 and Jurkat C8 ko cells ( E ) and Jurkat A3 and Jurkat RIPK1 ko cells ( F ) were pretreated for 1 h with 5 µM BV6, 50 µM zVAD-fmk and 10 µM Nec-1s and subsequently treated with 500 ng/ml CD95L for indicated timepoints ( B , C ), 2 h ( C ) or 5 h ( A , E , F ). pRIPK1- and RIPK1-IPs were carried out using anti-pRIPK1 and anti-RIPK1 antibodies, respectively, and analyzed by western blot. Actin was used as a loading control for the total cellular lysates (input). “Beads-only” pulldown (BC) was used as a negative control for IPs. One representative experiment out of three ( A , B , D – F ) or two ( C ) is shown. s.e.-short exposure, l.e. long exposure, IP immunoprecipitation, BC beads-only control pulldown, * unspecific band, IgG H the heavy chain of antibody. .

Article Snippet: The RIPK1 inhibitor 7-Cl-O-Nec-1 (Nec-1s; 5.04297.0001) and the RIPK3 inhibitor GSK872 (5.30389) were obtained from Merck, Germany.

Techniques: Western Blot, Control, Negative Control, Immunoprecipitation

$( A – C ) SUIT-020 cells were prestimulated with 5 µM BV6 and 50 µM zVAD-fmk for 1 h and afterwards stimulated with 500 ng/mL CD95L for 2 h. Total cellular lysate was fractionated by gel filtration. The different fractions were pooled (2–6; 7–10, 11–15) followed by casp8-IP ( A ), FADD-IP ( B ) or RIPK1-IP ( C ). Total cellular lysates and IPs were analyzed by western blot and probed for the indicated proteins. Actin was used as a loading control for total cellular lysates (input). One representative experiment out of two is shown. ( D ) Groups of proteins identified by mass spectrometry analysis were analyzed by GO bioinformatics analysis. IPs from CBZ-treated cells (FADD-IP/CBZ, FLIP-IP/CBZ, pRIPK1-IP/CBZ) were analyzed against IPs from untreated cells (FADD-IP/control, FLIP-IP/control, pRIPK-IP/control and beads-only control). The major groups of proteins are shown. The full analysis is presented in Dataset EV . The lowest significance is shown in black, the highest in light yellow. The proteins from the necroptotic cell death group have a similar significance to the proteins from the myosin group and immunoglobulin, which always have a high abundance in the IP experiments. ( E , F ) SUIT-020 cells were prestimulated with 5 µM BV6 and 50 µM zVAD-fmk for 1 h and afterwards stimulated with 500 ng/mL CD95L for 2 h. IP was done using anti-pRIPK1 ( E ) or anti-FADD ( F ) antibodies. The IPs were analyzed by AQUA peptide-based mass spectrometry analysis. The amounts of the AQUA peptides (fmols) corresponding to each protein, that were detected in the IP, are shown. Mean and standard deviations from three experiments are shown. Box plots show the distribution of the data using the median (center line), interquartile range (IQR; box limits represent the first and third quartiles, Q1 and Q3) and whiskers extending from the lower and upper quartiles to 1.5 × IQR. Points outside the whiskers are considered outliers and are shown as single points. ( G ) Gene expression levels in cancer tissues, as obtained from the TCGA data portal ( https://www.cancer.gov/tcga ). Gene expression levels were obtained using the RNA-Seq technique. IP immunoprecipitation, CBZ CD95L/BV6/zVAD-fmk, s.e. short exposure, l.e. long exposure, * unspecific band, IgG L the light chain of antibody. .$

Journal: The EMBO Journal

Article Title: Oligomerised RIPK1 is the main core component of the CD95 necrosome

doi: 10.1038/s44318-025-00433-0

Figure Lengend Snippet: ( A – C ) SUIT-020 cells were prestimulated with 5 µM BV6 and 50 µM zVAD-fmk for 1 h and afterwards stimulated with 500 ng/mL CD95L for 2 h. Total cellular lysate was fractionated by gel filtration. The different fractions were pooled (2–6; 7–10, 11–15) followed by casp8-IP ( A ), FADD-IP ( B ) or RIPK1-IP ( C ). Total cellular lysates and IPs were analyzed by western blot and probed for the indicated proteins. Actin was used as a loading control for total cellular lysates (input). One representative experiment out of two is shown. ( D ) Groups of proteins identified by mass spectrometry analysis were analyzed by GO bioinformatics analysis. IPs from CBZ-treated cells (FADD-IP/CBZ, FLIP-IP/CBZ, pRIPK1-IP/CBZ) were analyzed against IPs from untreated cells (FADD-IP/control, FLIP-IP/control, pRIPK-IP/control and beads-only control). The major groups of proteins are shown. The full analysis is presented in Dataset EV . The lowest significance is shown in black, the highest in light yellow. The proteins from the necroptotic cell death group have a similar significance to the proteins from the myosin group and immunoglobulin, which always have a high abundance in the IP experiments. ( E , F ) SUIT-020 cells were prestimulated with 5 µM BV6 and 50 µM zVAD-fmk for 1 h and afterwards stimulated with 500 ng/mL CD95L for 2 h. IP was done using anti-pRIPK1 ( E ) or anti-FADD ( F ) antibodies. The IPs were analyzed by AQUA peptide-based mass spectrometry analysis. The amounts of the AQUA peptides (fmols) corresponding to each protein, that were detected in the IP, are shown. Mean and standard deviations from three experiments are shown. Box plots show the distribution of the data using the median (center line), interquartile range (IQR; box limits represent the first and third quartiles, Q1 and Q3) and whiskers extending from the lower and upper quartiles to 1.5 × IQR. Points outside the whiskers are considered outliers and are shown as single points. ( G ) Gene expression levels in cancer tissues, as obtained from the TCGA data portal ( https://www.cancer.gov/tcga ). Gene expression levels were obtained using the RNA-Seq technique. IP immunoprecipitation, CBZ CD95L/BV6/zVAD-fmk, s.e. short exposure, l.e. long exposure, * unspecific band, IgG L the light chain of antibody. .

Article Snippet: The RIPK1 inhibitor 7-Cl-O-Nec-1 (Nec-1s; 5.04297.0001) and the RIPK3 inhibitor GSK872 (5.30389) were obtained from Merck, Germany.

Techniques: Filtration, Western Blot, Control, Mass Spectrometry, Gene Expression, RNA Sequencing, Immunoprecipitation

( A ) Comparison of RIPK1 and RIPK3 expression levels from normal and cancer pancreatic ductal adenocarcinoma. ( B ) HT29 cells were pretreated with 5 µM BV6 and 50 µM zVAD-fmk for 1 h. Afterwards cells were stimulated with 500 ng/mL CD95L for 5 h, which was followed by pRIPK1-IP, FADD-IP or c-FLIP-IP. The IPs were analyzed by mass spectrometry analysis. The results are presented in the Fig. . Box plots show the distribution of the data using the median (center line), interquartile range (IQR; box limits represent the first and third quartiles, Q1 and Q3) and whiskers extending from the lower and upper quartiles to 1.5 × IQR. Points outside the whiskers are considered outliers and are shown as single points. The average ratios of proteins were calculated using interquartile range and presented in this panel. ( C ) Molecular models of RIPK1 (DD-RHIM) predicted by AlphaFold3 for various oligomerization states, with models colored according to the confidence metric pLDDT. ( D ) Molecular models of RIPK1 (DD-RHIM) and FADD predicted by AlphaFold3. The heatmap on the right shows AlphaFold3 ipTM scores for different stoichiometries of FADD and RIPK1 proteins, with colors ranging from red (low confidence) to blue (high confidence). The molecular model of a 10 RIPK1/3 FADD complex, as predicted by AlphaFold3, is shown on the left.

Journal: The EMBO Journal

Article Title: Oligomerised RIPK1 is the main core component of the CD95 necrosome

doi: 10.1038/s44318-025-00433-0

Figure Lengend Snippet: ( A ) Comparison of RIPK1 and RIPK3 expression levels from normal and cancer pancreatic ductal adenocarcinoma. ( B ) HT29 cells were pretreated with 5 µM BV6 and 50 µM zVAD-fmk for 1 h. Afterwards cells were stimulated with 500 ng/mL CD95L for 5 h, which was followed by pRIPK1-IP, FADD-IP or c-FLIP-IP. The IPs were analyzed by mass spectrometry analysis. The results are presented in the Fig. . Box plots show the distribution of the data using the median (center line), interquartile range (IQR; box limits represent the first and third quartiles, Q1 and Q3) and whiskers extending from the lower and upper quartiles to 1.5 × IQR. Points outside the whiskers are considered outliers and are shown as single points. The average ratios of proteins were calculated using interquartile range and presented in this panel. ( C ) Molecular models of RIPK1 (DD-RHIM) predicted by AlphaFold3 for various oligomerization states, with models colored according to the confidence metric pLDDT. ( D ) Molecular models of RIPK1 (DD-RHIM) and FADD predicted by AlphaFold3. The heatmap on the right shows AlphaFold3 ipTM scores for different stoichiometries of FADD and RIPK1 proteins, with colors ranging from red (low confidence) to blue (high confidence). The molecular model of a 10 RIPK1/3 FADD complex, as predicted by AlphaFold3, is shown on the left.

Article Snippet: The RIPK1 inhibitor 7-Cl-O-Nec-1 (Nec-1s; 5.04297.0001) and the RIPK3 inhibitor GSK872 (5.30389) were obtained from Merck, Germany.

Techniques: Comparison, Expressing, Mass Spectrometry

( A ) The model confidence metrics, iPTM score for various models calculated for different ratios of RIPK1 and FADD proteins using AlphaFold2-multimer, are presented. The heatmap colors range from red (indicating low confidence) to blue (indicating high confidence). Models with the high iPTM score that fit to the experimentally determined stoichomteries obtained through AQUA peptides are indicated with *. ( B ) Schematic diagram of the RIPK1:FADD assembly is shown on the right generated by AlphaFold. Representative structural model of the RIPK1:FADD DD complex within the necrosome with high iPTM score is shown on the left. ( C ) Wild-type and mutant type-II DD interactions according to the necrosome structural model obtained by AlphaFold2-multimer are shown. The key residues are highlighted and their mutations are shown in red. Schematic representations of the DD interaction interfaces are displayed on the hexagon diagram. RIPK1 DD is shown in gold, FADD DD is shown in purple and CD95 DD is shown in green.

Journal: The EMBO Journal

Article Title: Oligomerised RIPK1 is the main core component of the CD95 necrosome

doi: 10.1038/s44318-025-00433-0

Figure Lengend Snippet: ( A ) The model confidence metrics, iPTM score for various models calculated for different ratios of RIPK1 and FADD proteins using AlphaFold2-multimer, are presented. The heatmap colors range from red (indicating low confidence) to blue (indicating high confidence). Models with the high iPTM score that fit to the experimentally determined stoichomteries obtained through AQUA peptides are indicated with *. ( B ) Schematic diagram of the RIPK1:FADD assembly is shown on the right generated by AlphaFold. Representative structural model of the RIPK1:FADD DD complex within the necrosome with high iPTM score is shown on the left. ( C ) Wild-type and mutant type-II DD interactions according to the necrosome structural model obtained by AlphaFold2-multimer are shown. The key residues are highlighted and their mutations are shown in red. Schematic representations of the DD interaction interfaces are displayed on the hexagon diagram. RIPK1 DD is shown in gold, FADD DD is shown in purple and CD95 DD is shown in green.

Article Snippet: The RIPK1 inhibitor 7-Cl-O-Nec-1 (Nec-1s; 5.04297.0001) and the RIPK3 inhibitor GSK872 (5.30389) were obtained from Merck, Germany.

Techniques: Generated, Mutagenesis

( A ) Model depicting the formation of the necrosome in response to CD95 stimulation. RIPK1 DD/RHIM is shown in gold, FADD DD is shown in purple and CD95 DD is shown in green. ( B ) Somatic Mutations Observed in Cancer Tissues Involving the RIPK1 DD. Somatic mutations affecting the DD of RIPK1, including K599R and others, were extracted from the COSMIC database (Tate et al, ). Residues with somatic mutations observed located on type-II DD protein–protein interaction interfaces are indicated in cyan. RIPK1 DD is shown in gold, FADD DD in purple. Residues corresponding to the potential ubiquitinylation sites are denoted and colored in black. The scheme depicting the predicted functional significance of residues R599 and R603 in the homotypic type-I interaction of RIPK1 DD is shown on the right.

Journal: The EMBO Journal

Article Title: Oligomerised RIPK1 is the main core component of the CD95 necrosome

doi: 10.1038/s44318-025-00433-0

Figure Lengend Snippet: ( A ) Model depicting the formation of the necrosome in response to CD95 stimulation. RIPK1 DD/RHIM is shown in gold, FADD DD is shown in purple and CD95 DD is shown in green. ( B ) Somatic Mutations Observed in Cancer Tissues Involving the RIPK1 DD. Somatic mutations affecting the DD of RIPK1, including K599R and others, were extracted from the COSMIC database (Tate et al, ). Residues with somatic mutations observed located on type-II DD protein–protein interaction interfaces are indicated in cyan. RIPK1 DD is shown in gold, FADD DD in purple. Residues corresponding to the potential ubiquitinylation sites are denoted and colored in black. The scheme depicting the predicted functional significance of residues R599 and R603 in the homotypic type-I interaction of RIPK1 DD is shown on the right.

Article Snippet: The RIPK1 inhibitor 7-Cl-O-Nec-1 (Nec-1s; 5.04297.0001) and the RIPK3 inhibitor GSK872 (5.30389) were obtained from Merck, Germany.

Techniques: Functional Assay

( A ) Somatic Mutations Observed in Cancer Tissues within RIPK1 DD. DD interaction interfaces of RIPK1 are highlighted with 1 (type-I), 2 (type-II) or 3 (type-III) underlinings. Mutations were extracted from the COSMIC database. ( B ) Jurkat A3 and Jurkat RIPK1 ko cells were pretreated with 5 µM BV6, 50 µM zVAD-fmk and 10 µM Nec-1s for 1 h and afterwards stimulated with 50 ng/mL CD95L for 24 h. The complete experiment with all controls is shown in Fig. . ( C ) Jurkat RIPK1 ko cells were transfected with empty vector, WT-RIPK1 or RIPK1-AD (M637A, I641D). Transfected cells were pretreated with 5 µM BV6, 50 µM zVAD-fmk and 10 µM Nec-1s for 1 h and afterwards stimulated with 50 ng/mL CD95L for 24 h. Western blot transfection control is shown. Jurkat A3 cells were loaded on the same gel to have a positive control for RIPK1 expression (RIP CTR). Actin served as loading control. ( D ) Jurkat FADD ko cells were transfected with empty vector, WT-FADD, FADD-DRS (L172D, D175R, L176S) or FADD-ADRN (M170A, L172D, D175R, L176N). Transfected cells were pretreated with 5 µM BV6 and 50 µM zVAD-fmk for 1 h and afterward stimulated with 500 ng/mL CD95L for 24 h. Western blot transfection control is shown. Actin served as loading control. The mean and standard deviation from three experiments are shown. Cell viability was measured using the Cell Titer-Glo®-Luminescent Cell Viability Assay by Promega. Untreated cells were taken as 100%. Statistics were calculated with unpaired one-way ANOVA with Tukey post hoc test to compare two conditions. Significance values: **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05; ns not significant. P values from left to right for ( B ) P = 0.5607, P = 0.0047, P > 0.9999, ( C ) P = 0.0018, P < 0.0001, ( D ) P < 0.0001, P > 0.9999, P > 0.9999, P < 0.0001, P < 0.0001, P = 0.0330, P < 0.0001, P = 0.0012. wt wild type, - untransfected, ø empty vector. .

Journal: The EMBO Journal

Article Title: Oligomerised RIPK1 is the main core component of the CD95 necrosome

doi: 10.1038/s44318-025-00433-0

Figure Lengend Snippet: ( A ) Somatic Mutations Observed in Cancer Tissues within RIPK1 DD. DD interaction interfaces of RIPK1 are highlighted with 1 (type-I), 2 (type-II) or 3 (type-III) underlinings. Mutations were extracted from the COSMIC database. ( B ) Jurkat A3 and Jurkat RIPK1 ko cells were pretreated with 5 µM BV6, 50 µM zVAD-fmk and 10 µM Nec-1s for 1 h and afterwards stimulated with 50 ng/mL CD95L for 24 h. The complete experiment with all controls is shown in Fig. . ( C ) Jurkat RIPK1 ko cells were transfected with empty vector, WT-RIPK1 or RIPK1-AD (M637A, I641D). Transfected cells were pretreated with 5 µM BV6, 50 µM zVAD-fmk and 10 µM Nec-1s for 1 h and afterwards stimulated with 50 ng/mL CD95L for 24 h. Western blot transfection control is shown. Jurkat A3 cells were loaded on the same gel to have a positive control for RIPK1 expression (RIP CTR). Actin served as loading control. ( D ) Jurkat FADD ko cells were transfected with empty vector, WT-FADD, FADD-DRS (L172D, D175R, L176S) or FADD-ADRN (M170A, L172D, D175R, L176N). Transfected cells were pretreated with 5 µM BV6 and 50 µM zVAD-fmk for 1 h and afterward stimulated with 500 ng/mL CD95L for 24 h. Western blot transfection control is shown. Actin served as loading control. The mean and standard deviation from three experiments are shown. Cell viability was measured using the Cell Titer-Glo®-Luminescent Cell Viability Assay by Promega. Untreated cells were taken as 100%. Statistics were calculated with unpaired one-way ANOVA with Tukey post hoc test to compare two conditions. Significance values: **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05; ns not significant. P values from left to right for ( B ) P = 0.5607, P = 0.0047, P > 0.9999, ( C ) P = 0.0018, P < 0.0001, ( D ) P < 0.0001, P > 0.9999, P > 0.9999, P < 0.0001, P < 0.0001, P = 0.0330, P < 0.0001, P = 0.0012. wt wild type, - untransfected, ø empty vector. .

Article Snippet: The RIPK1 inhibitor 7-Cl-O-Nec-1 (Nec-1s; 5.04297.0001) and the RIPK3 inhibitor GSK872 (5.30389) were obtained from Merck, Germany.

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Positive Control, Expressing, Standard Deviation, Cell Viability Assay

( A ) Jurkat A3 and Jurkat A3 RIPK1 ko cells were treated with indicated concentrations of CD95L for 24 h. ( B , C ) Jurkat A3 ( B ) or Jurkat A3 RIPK1 ko ( C ) cells were prestimulated with 5 µM BV6, 50 µM zVAD-fmk and 10 µM Nec-1s for 1 h and afterwards stimulated with 50 ng/mL CD95L for 24 h. Cell viability was measured using the Cell Titer-Glo®-Luminescent Cell Viability Assay by Promega. The cell viability measurements of untreated cells were taken as 100%. Mean and standard deviation from three independent experiments are shown. Statistics were calculated with unpaired one-way ANOVA with Tukey post hoc test to compare two conditions. Significance values: **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05; ns not significant. ( D , E ) HeLa FADD ko RIPK3 cells were transfected with empty vector, WT-FADD, FADD-DRS (L172D, D175R, L176S) or FADD-ADRN (M170A, L172D, D175R, L176N). Transfected cells were pretreated with 5 µM BV6, 50 µM zVAD-fmk or 10 µM Nec-1s for 1 h and afterwards stimulated with 500 or 1000 ng/mL CD95L for 3 h or as indicated. ( F , G ) HeLa RIPK1 ko RIPK3 cells were transfected with empty vector, WT-RIPK1 or RIPK1-AD (M637A, I641D). Transfected cells were pretreated with 5 µM BV6 and 50 µM zVAD-fmk for 1 h and afterwards stimulated with 500 or 1000 ng/mL CD95L for 3 h. Total cellular lysates were analyzed using western blot with the indicated antibodies. Actin served as loading control. One representative western blot out of three is shown. WT wild type, - untransfected, ø empty vector, s.e. short exposure, l.e. long exposure. .

Journal: The EMBO Journal

Article Title: Oligomerised RIPK1 is the main core component of the CD95 necrosome

doi: 10.1038/s44318-025-00433-0

Figure Lengend Snippet: ( A ) Jurkat A3 and Jurkat A3 RIPK1 ko cells were treated with indicated concentrations of CD95L for 24 h. ( B , C ) Jurkat A3 ( B ) or Jurkat A3 RIPK1 ko ( C ) cells were prestimulated with 5 µM BV6, 50 µM zVAD-fmk and 10 µM Nec-1s for 1 h and afterwards stimulated with 50 ng/mL CD95L for 24 h. Cell viability was measured using the Cell Titer-Glo®-Luminescent Cell Viability Assay by Promega. The cell viability measurements of untreated cells were taken as 100%. Mean and standard deviation from three independent experiments are shown. Statistics were calculated with unpaired one-way ANOVA with Tukey post hoc test to compare two conditions. Significance values: **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05; ns not significant. ( D , E ) HeLa FADD ko RIPK3 cells were transfected with empty vector, WT-FADD, FADD-DRS (L172D, D175R, L176S) or FADD-ADRN (M170A, L172D, D175R, L176N). Transfected cells were pretreated with 5 µM BV6, 50 µM zVAD-fmk or 10 µM Nec-1s for 1 h and afterwards stimulated with 500 or 1000 ng/mL CD95L for 3 h or as indicated. ( F , G ) HeLa RIPK1 ko RIPK3 cells were transfected with empty vector, WT-RIPK1 or RIPK1-AD (M637A, I641D). Transfected cells were pretreated with 5 µM BV6 and 50 µM zVAD-fmk for 1 h and afterwards stimulated with 500 or 1000 ng/mL CD95L for 3 h. Total cellular lysates were analyzed using western blot with the indicated antibodies. Actin served as loading control. One representative western blot out of three is shown. WT wild type, - untransfected, ø empty vector, s.e. short exposure, l.e. long exposure. .

Article Snippet: The RIPK1 inhibitor 7-Cl-O-Nec-1 (Nec-1s; 5.04297.0001) and the RIPK3 inhibitor GSK872 (5.30389) were obtained from Merck, Germany.

Techniques: Cell Viability Assay, Standard Deviation, Transfection, Plasmid Preparation, Western Blot, Control

( A , B ) HeLa FADD ko RIPK3 cells were transfected with empty vector, WT-FADD, FADD-DRS (L172D, D175R, L176S) or FADD-ADRN (M170A, L172D, D175R, L176N). Transfected cells were pretreated with 5 µM BV6, 50 µM zVAD-fmk or 10 µM Nec-1s for 1 h and afterwards stimulated with 500 ng/mL CD95L for 8 h ( A ) or 24 h ( B ). ( C , D ) HeLa RIPK1 ko RIPK3 cells were transfected with empty vector, WT-RIPK1 or RIPK1-AD (M637A, I641D). Transfected cells were pretreated with 5 µM BV6, 50 µM zVAD-fmk or 10 µM Nec-1s for 1 h and afterwards stimulated with 500 ng/mL CD95L for 8 h ( C ) or 24 h ( D ). Transfection controls are included in (EV9D-G). The mean and standard deviation from the three experiments are shown. Cell viability was measured using the Cell Titer-Glo®-Luminescent Cell Viability Assay by Promega. Untreated cells were taken as 100%. Statistics were calculated with unpaired one-way ANOVA with Tukey post hoc test to compare two conditions. Significance values: **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05; ns not significant. P values from left to right for ( A ) P = 0.0051, P = 0.2633, P = 0.0050, ( B ) P = 0.0037, P = 0.0272, P = 0.0024, ( C ) P = 0.0002, P = 0.0003, ( D ) P < 0.0001, P < 0.0001. ( E , F ) The AlphaFold3-generated model includes a DD oligomer formed by the RIPK1 DD (gold) and FADD DD (purple), a DED filament composed of FADD-DED (brown), full-length procaspase-8 (blue), and c-FLIP L (yellow), as well as a RHIM oligomer consisting of RIPK1 and RIPK3 RHIM subunits. This complex comprises 2× c-FLIP L , 4× procaspase-8, 3× FADD, 7× RIPK1, and 1 RIPK3 proteins. The RHIM oligomer is highlighted with the molecular surface. The side view is shown on the left ( E ) and top view is shown on the right ( F ). .

Journal: The EMBO Journal

Article Title: Oligomerised RIPK1 is the main core component of the CD95 necrosome

doi: 10.1038/s44318-025-00433-0

Figure Lengend Snippet: ( A , B ) HeLa FADD ko RIPK3 cells were transfected with empty vector, WT-FADD, FADD-DRS (L172D, D175R, L176S) or FADD-ADRN (M170A, L172D, D175R, L176N). Transfected cells were pretreated with 5 µM BV6, 50 µM zVAD-fmk or 10 µM Nec-1s for 1 h and afterwards stimulated with 500 ng/mL CD95L for 8 h ( A ) or 24 h ( B ). ( C , D ) HeLa RIPK1 ko RIPK3 cells were transfected with empty vector, WT-RIPK1 or RIPK1-AD (M637A, I641D). Transfected cells were pretreated with 5 µM BV6, 50 µM zVAD-fmk or 10 µM Nec-1s for 1 h and afterwards stimulated with 500 ng/mL CD95L for 8 h ( C ) or 24 h ( D ). Transfection controls are included in (EV9D-G). The mean and standard deviation from the three experiments are shown. Cell viability was measured using the Cell Titer-Glo®-Luminescent Cell Viability Assay by Promega. Untreated cells were taken as 100%. Statistics were calculated with unpaired one-way ANOVA with Tukey post hoc test to compare two conditions. Significance values: **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05; ns not significant. P values from left to right for ( A ) P = 0.0051, P = 0.2633, P = 0.0050, ( B ) P = 0.0037, P = 0.0272, P = 0.0024, ( C ) P = 0.0002, P = 0.0003, ( D ) P < 0.0001, P < 0.0001. ( E , F ) The AlphaFold3-generated model includes a DD oligomer formed by the RIPK1 DD (gold) and FADD DD (purple), a DED filament composed of FADD-DED (brown), full-length procaspase-8 (blue), and c-FLIP L (yellow), as well as a RHIM oligomer consisting of RIPK1 and RIPK3 RHIM subunits. This complex comprises 2× c-FLIP L , 4× procaspase-8, 3× FADD, 7× RIPK1, and 1 RIPK3 proteins. The RHIM oligomer is highlighted with the molecular surface. The side view is shown on the left ( E ) and top view is shown on the right ( F ). .

Article Snippet: The RIPK1 inhibitor 7-Cl-O-Nec-1 (Nec-1s; 5.04297.0001) and the RIPK3 inhibitor GSK872 (5.30389) were obtained from Merck, Germany.

Techniques: Transfection, Plasmid Preparation, Standard Deviation, Cell Viability Assay, Generated

This model includes trimerization of the receptor upon CD95L binding, formation of the putative intermediate complex involving CD95/RIPK1/FADD DD interactions, leading to assembly of complex II or DISC, promoting necroptosis or apoptosis, respectively. The designations are shown at the bottom of the figure. According to the model, a high concentration of deubiquitinated RIPK1-forming oligomers is required to initiate necrosome assembly. In addition, FADD DD drives the initial assembly of complex II via interactions of FADD type-II DD interface and with type-II DD interface of RIPK1.

Journal: The EMBO Journal

Article Title: Oligomerised RIPK1 is the main core component of the CD95 necrosome

doi: 10.1038/s44318-025-00433-0

Figure Lengend Snippet: This model includes trimerization of the receptor upon CD95L binding, formation of the putative intermediate complex involving CD95/RIPK1/FADD DD interactions, leading to assembly of complex II or DISC, promoting necroptosis or apoptosis, respectively. The designations are shown at the bottom of the figure. According to the model, a high concentration of deubiquitinated RIPK1-forming oligomers is required to initiate necrosome assembly. In addition, FADD DD drives the initial assembly of complex II via interactions of FADD type-II DD interface and with type-II DD interface of RIPK1.

Article Snippet: The RIPK1 inhibitor 7-Cl-O-Nec-1 (Nec-1s; 5.04297.0001) and the RIPK3 inhibitor GSK872 (5.30389) were obtained from Merck, Germany.

Techniques: Binding Assay, Concentration Assay

(A) Immunoblot analysis of RagA-deficient iBMDM clonal cell lines. (B–E) RagA-deficient iBMDM or non-target sgRNA (WT) control cells were treated with LPS (1 μg/mL) alone or co-treated with LPS (1 μg/mL) and the TAK1 inhibitor 5z7 (250 nM) for 3 h. PI fluorescence and LDH release were quantified as a measure of membrane disruption/lysis (B and C). GSDMD processing and oligomerization were assessed by immunoblot under reducing (D) or non-reducing (E) conditions. (F) Immunoblot analysis of RagA-, caspase-8-, or RIPK1-deficient iBMDMs expressing NT-GSDMD under Dox-inducible promoter. (G and H) RagA-, caspase-8-, or RIPK1-deficient iBMDMs expressing NT-GSDMD under Dox-inducible promoter were treated with LPS (1 μg/mL) alone or co-treated with LPS and the TAK1 inhibitor 5z7 (250 nM) for 6 h. PI fluorescence and LDH release were quantified as measure of membrane disruption/lysis. (I–K) RagA-, caspase-8-, or RIPK1-deficient iBMDMs iBMDMs expressing NT-GSDMD under Dox-inducible promoter were treated with Dox (0.5 μg/mL) for 16 h. PI fluorescence and LDH release were quantified as measure of membrane disruption/lysis. Fraction of BFP-positive cells was measured by flow cytometry. Cells indicated as WT were electroporated with a non-targeting sgRNA. Data are mean ± SEM of three experiments. Immunoblots show representative result of three repeats. Statistical significance was determined by two-way ANOVA: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Journal: Cell reports

Article Title: Gasdermin D pore-forming activity is redox-sensitive

doi: 10.1016/j.celrep.2023.112008

Figure Lengend Snippet: (A) Immunoblot analysis of RagA-deficient iBMDM clonal cell lines. (B–E) RagA-deficient iBMDM or non-target sgRNA (WT) control cells were treated with LPS (1 μg/mL) alone or co-treated with LPS (1 μg/mL) and the TAK1 inhibitor 5z7 (250 nM) for 3 h. PI fluorescence and LDH release were quantified as a measure of membrane disruption/lysis (B and C). GSDMD processing and oligomerization were assessed by immunoblot under reducing (D) or non-reducing (E) conditions. (F) Immunoblot analysis of RagA-, caspase-8-, or RIPK1-deficient iBMDMs expressing NT-GSDMD under Dox-inducible promoter. (G and H) RagA-, caspase-8-, or RIPK1-deficient iBMDMs expressing NT-GSDMD under Dox-inducible promoter were treated with LPS (1 μg/mL) alone or co-treated with LPS and the TAK1 inhibitor 5z7 (250 nM) for 6 h. PI fluorescence and LDH release were quantified as measure of membrane disruption/lysis. (I–K) RagA-, caspase-8-, or RIPK1-deficient iBMDMs iBMDMs expressing NT-GSDMD under Dox-inducible promoter were treated with Dox (0.5 μg/mL) for 16 h. PI fluorescence and LDH release were quantified as measure of membrane disruption/lysis. Fraction of BFP-positive cells was measured by flow cytometry. Cells indicated as WT were electroporated with a non-targeting sgRNA. Data are mean ± SEM of three experiments. Immunoblots show representative result of three repeats. Statistical significance was determined by two-way ANOVA: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: RIPK1 inhibitor (7-Cl-O-Nec-1) and RIPK3 inhibitor (GSK’872) were both from MilliporeSigma.

Techniques: Western Blot, Control, Fluorescence, Membrane, Disruption, Lysis, Expressing, Flow Cytometry

Journal: Cell reports

Article Title: Gasdermin D pore-forming activity is redox-sensitive

doi: 10.1016/j.celrep.2023.112008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: RIPK1 inhibitor (7-Cl-O-Nec-1) and RIPK3 inhibitor (GSK’872) were both from MilliporeSigma.

Techniques: Virus, Recombinant, Protease Inhibitor, Sterility, Binding Assay, Saline, Plasmid Preparation, Transfection, CyQUANT Assay, LDH Cytotoxicity Assay, Bicinchoninic Acid Protein Assay, Mutagenesis, Retroviral, Software, Live Cell Imaging, Laser-Scanning Microscopy, Imaging, Mass Spectrometry

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