p rip1  (Cell Signaling Technology Inc)

Journal: Brain and Behavior

Article Title: Study on the Function and Mechanism of Neutrophil Extracellular Traps in Regulating Necroptosis Following Traumatic Brain Injury

doi: 10.1002/brb3.71275

Figure Lengend Snippet: Effects of Cl‐amidine and DNase I on necroptosis‐related protein expression after TBI. (A) Western blot analysis of RIP1, RIP3, MLKL, P‐RIP1, P‐RIP3, and P‐MLKL protein levels in the cortex of mice from Sham, TBI+Vehicle, TBI+Cl‐amidine, and TBI+DNase I groups. GAPDH was used as the loading control ( n = 4 per group). (B–G) Quantification of relative protein levels of RIP1, RIP3, MLKL, P‐RIP1, P‐RIP3, and P‐MLKL, normalized to GAPDH. Data are shown as mean ± SD ( n = 4 per group). Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the indicated groups; ns: not significant.

Article Snippet: The membranes were blocked with 5% skimmed milk prepared in TBST (Tris‐buffered saline with 0.1% Tween‐20) at room temperature for 1 h. They were then incubated overnight at 4°C with the following primary antibodies: PAD4 (1:1000, 214810, Abcam), MPO (1:1000, ab208670, Abcam), Bcl‐2 (1:1000, A0208, Abclonal), Bax (1:1000, A19684, Abclonal), RIP1 (1:1000, #3493, Cell Signaling Technology), RIP3 (1:1000, #95702, Cell Signaling Technology), MLKL (1:1000, #37705, Cell Signaling Technology), P‐RIP1 (1:1000, #31122, Cell Signaling Technology), P‐RIP3 (1:1000, #91702, Cell Signaling Technology), P‐MLKL (1:1000, #37333, Cell Signaling Technology), and GAPDH (1:1000, AB‐P‐R001, GOODHERE Biotech).

Techniques: Expressing, Western Blot, Control

Journal: Nature Communications

Article Title: Decoding necrosome assembly: harmonizing signal amplification and attenuation through optimal RIP3 stoichiometry

doi: 10.1038/s41467-025-67098-5

Figure Lengend Snippet: a Schematic of TNF-induced supramolecular signaling leading to necrosome formation. b Immunoblot of the indicated proteins in lysates from RIP3-expressing HeLa cells, untreated or treated with TSZ (TNF + Smac mimetic + zVAD-fmk) for 4 h. LE, long exposure. c Confocal images of MLKL -knockout (KO) HeLa cells reconstituted with HA-RIP3 and MLKL-Flag and stimulated with DMSO or TSZ for 3 h. Fixed cells were immunolabeled with antibodies against RIP1 (green), HA (magenta), and Flag (yellow). DNA was counterstained with Hoechst 33342 (blue). Enlarged regions (white boxes) depict cytosolic necrosomes (red arrows), demonstrating co-localization of all three proteins. Data in ( b , c ) are representative of two and three independent experiments, respectively. Scale bars: 10 μm (overviews) and 2 μm (insets). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used throughout this report: anti-RIP1 (Cell Signaling, 3493, 1:150 for immunofluorescence (IF), 1:1,000 for western blotting (WB)), anti-phospho Ser166 RIP1 (Cell Signaling, 65746, 1:1,000 for WB), anti-RIP3 (Cell Signaling, 13526, 1:1,000 for WB), anti-phospho Ser227 RIP3 (Abcam, ab209384, 1:1,000 for WB), anti-MLKL (Abcam, ab184718, 1:200 for IF; 1:1,000 for WB), anti-phospho Ser358 MLKL (Abcam, ab187091, 1:1,000 for WB), anti-HA (Santa Cruz, sc-7392, 1:200 for IF, 1:1,000 for WB; Cell Signaling, 3724, 1:200 for IF, 1:1,000 for WB; ABclonal, AE008, 1:200 for IF), anti-Flag (Abmart, M20008L, 1:1000 for IF, 1:5,000 for WB; Biolegend, 637301, 1:200 for IF), anti-GAPDH (Proteintech, 60004-1-Ig, 1:5,000 for WB), goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to Alexa Fluor 488, 568 or 647 (Thermo Fisher Scientific, A11034, A11004 or A21247, 1:1,000 for IF) and goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to CF 488 A, CF568 or CF 647 (Biotium, 20015, 20800, 20801, 20808 or 20809, 1:500 for IF).

Techniques: Western Blot, Expressing, Knock-Out, Immunolabeling

Journal: Nature Communications

Article Title: Decoding necrosome assembly: harmonizing signal amplification and attenuation through optimal RIP3 stoichiometry

doi: 10.1038/s41467-025-67098-5

Figure Lengend Snippet: a STORM workflow and image analysis pipeline. Detailed procedures for image acquisition and segmentation are described in the Methods. b Single-color STORM images of RIP1, RIP3, and MLKL in HA-RIP3-expressing HeLa cells treated with TSZ for the indicated durations. Insets show magnified views (white boxes) of RIP1, RIP3, or MLKL within necrosomes. Images are representative of two independent experiments per condition. c Area distributions of RIP1-, RIP3-, and MLKL-stained rods in HA-RIP3-expressing HeLa cells treated with TSZ for 2 h ( n = 53 RIP1, 114 RIP3, 63 MLKL) or 4 h ( n = 77 RIP1, 168 RIP3, 37 MLKL). d Proportions of round- versus rod-shaped RIP1, RIP3, and MLKL structures in Flag-RIP3–expressing HeLa cells treated with TSZ for 2 h or 4 h, classified based on STORM images. e Scheme of quantitative mass spectrometry (MS) workflow. RIP3 and MLKL complexes were immunoprecipitated with anti-Flag agarose beads. IP samples were digested with trypsin, and peptides were analyzed using DDA and SWATH-MS. Details are described in the Methods section. Panels ( a , e ) were created with BioRender.com ( https://BioRender.com/cril410 ). f Ratios of caspase-8, MLKL, and RIP3 to RIP1 in Flag-RIP3/Flag-MLKL-enriched necrosomes, as determined by MS. Data are presented as mean ± SD from three biological replicates per group. g Proposed model of the modular and cooperative architecture of the necrosome. Stoichiometric ratios (RIP1:RIP3:MLKL:Caspase-8 ≈ 2:6:4:1) were derived from IP-MS analysis. Published (PDB) and predicted (AlphaFold) structural models illustrate domain-specific interactions: DD oligomerization (TNFR, TRADD, RIP1), RHIM-driven hetero-/homo-amyloid assembly (RIP1–RIP3), and downstream effector recruitment (FADD–caspase-8; RIP3–MLKL). Scale bars: 5 μm (overviews) and 100 nm (insets). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used throughout this report: anti-RIP1 (Cell Signaling, 3493, 1:150 for immunofluorescence (IF), 1:1,000 for western blotting (WB)), anti-phospho Ser166 RIP1 (Cell Signaling, 65746, 1:1,000 for WB), anti-RIP3 (Cell Signaling, 13526, 1:1,000 for WB), anti-phospho Ser227 RIP3 (Abcam, ab209384, 1:1,000 for WB), anti-MLKL (Abcam, ab184718, 1:200 for IF; 1:1,000 for WB), anti-phospho Ser358 MLKL (Abcam, ab187091, 1:1,000 for WB), anti-HA (Santa Cruz, sc-7392, 1:200 for IF, 1:1,000 for WB; Cell Signaling, 3724, 1:200 for IF, 1:1,000 for WB; ABclonal, AE008, 1:200 for IF), anti-Flag (Abmart, M20008L, 1:1000 for IF, 1:5,000 for WB; Biolegend, 637301, 1:200 for IF), anti-GAPDH (Proteintech, 60004-1-Ig, 1:5,000 for WB), goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to Alexa Fluor 488, 568 or 647 (Thermo Fisher Scientific, A11034, A11004 or A21247, 1:1,000 for IF) and goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to CF 488 A, CF568 or CF 647 (Biotium, 20015, 20800, 20801, 20808 or 20809, 1:500 for IF).

Techniques: Expressing, Staining, Mass Spectrometry, Immunoprecipitation, Data-independent acquisition, Derivative Assay, Protein-Protein interactions

Journal: Nature Communications

Article Title: Decoding necrosome assembly: harmonizing signal amplification and attenuation through optimal RIP3 stoichiometry

doi: 10.1038/s41467-025-67098-5

Figure Lengend Snippet: a Diagram illustrating a basic model depicting higher-order complexes assembly by two components. b Effects of varying the number of B (n) or A (m) molecules on output, with one component fixed at 1 within the subunit. Green arrows indicate changes in output signal and regulatory behavior with increasing input intensity as n or m varies. c Impact of diverse assembly stoichiometries on output (upper panel) and regulatory behavior (lower panel). The sensitivity coefficient quantifies the threshold response by measuring the reduction in stimulus intensity required to decrease the output from its maximum (20 a.u.). d Schematic of necrosome assembly in MLKL -KO HeLa cells. Immunoblots are representative of three independent experiments. p-RIP3 was quantified by band intensity. The kinetic model (Supplementary Table ) recapitulates experimental RIP3 phosphorylation dynamics. e Model predictions of how RIP3:RIP1 stoichiometries in necrosomes affect necroptotic signal output. f Predicted effect of reduced RIP1 expression on RIP3 phosphorylation (upper panel), alongside experimental validation using literature data (lower panel). p-RIP3 was quantified by band intensity and RIP1 levels were controlled by shRNA-mediated knockdown. g Schematic of MAP7-based calibration system created in BioRender ( https://BioRender.com/zaak4bk ).HeLa cells expressing Flag-MAP7-HA or Flag-MAP7-P2A-Flag-MAP7-HA (“2Flag-MAP7-HA”) are expected to display Flag:HA stoichiometries of 1:1 and 2:1, respectively. Dual-color confocal ( h ) and STORM ( j ) images of HeLa cells expressing Flag-MAP7-HA or 2Flag-MAP7-HA, immunolabeled with anti-Flag (purple) and anti-HA (green) antibodies. Quantification of fluorescence intensity ( i ; n = 40 fields per group) and localization counts ( k ; n = 45 structures per group) confirmed Flag:HA ratios matched expected values. Insets show dual-labeled tubulin with calculated Flag:HA ratios. l Three-color STORM of necrosomes in HA-RIP1/Flag-RIP3–expressing RIP1-MLKL -DKO HeLa cells treated with TSZ for 4 h, labeled for HA-RIP1 (green), Flag-RIP3 (purple), and p-RIP3 (yellow). Insets highlight necrosomes with calculated Flag:HA ratios and p-RIP3 localizations. m Quantification of RIP3:RIP1 stoichiometry and relative p-RIP3 levels (localizations) in individual necrosomes ( n = 89). Images in ( h , j , l ) are representative of two independent experiments. Scale bars: 10 μm ( h , overviews in j and l ) and 300 nm (insets in j and l ). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used throughout this report: anti-RIP1 (Cell Signaling, 3493, 1:150 for immunofluorescence (IF), 1:1,000 for western blotting (WB)), anti-phospho Ser166 RIP1 (Cell Signaling, 65746, 1:1,000 for WB), anti-RIP3 (Cell Signaling, 13526, 1:1,000 for WB), anti-phospho Ser227 RIP3 (Abcam, ab209384, 1:1,000 for WB), anti-MLKL (Abcam, ab184718, 1:200 for IF; 1:1,000 for WB), anti-phospho Ser358 MLKL (Abcam, ab187091, 1:1,000 for WB), anti-HA (Santa Cruz, sc-7392, 1:200 for IF, 1:1,000 for WB; Cell Signaling, 3724, 1:200 for IF, 1:1,000 for WB; ABclonal, AE008, 1:200 for IF), anti-Flag (Abmart, M20008L, 1:1000 for IF, 1:5,000 for WB; Biolegend, 637301, 1:200 for IF), anti-GAPDH (Proteintech, 60004-1-Ig, 1:5,000 for WB), goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to Alexa Fluor 488, 568 or 647 (Thermo Fisher Scientific, A11034, A11004 or A21247, 1:1,000 for IF) and goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to CF 488 A, CF568 or CF 647 (Biotium, 20015, 20800, 20801, 20808 or 20809, 1:500 for IF).

Techniques: Western Blot, Phospho-proteomics, Expressing, Biomarker Discovery, shRNA, Knockdown, Immunolabeling, Fluorescence, Labeling

Journal: Nature Communications

Article Title: Decoding necrosome assembly: harmonizing signal amplification and attenuation through optimal RIP3 stoichiometry

doi: 10.1038/s41467-025-67098-5

Figure Lengend Snippet: a Diagram illustrating model of higher-order complexes formed by three components. b Impact of diverse assembly configurations of A, B and C on output. c Intuitive explanation of the optimal configuration for the midstream signal. The three-component complex can be divided into two assemblies: TCS-1, where B’s higher-order assembly amplifies the signal from A; and TCS-2, where B inhibits signal transmission to C. d Impact of input intensity and initial levels of A, B, and C on B’s optimal assembly configuration. e Multi-color STORM images of MLKL -KO HeLa cells expressing HA-RIP3 and MLKL-Flag after TSZ treatment, labeled for RIP1 (green), HA-RIP3 (purple) and MLKL-Flag (yellow). Images are representative of three independent experiments. f Schematic of TNF-induced RIP1-RIP3-MLKL necrosomes assembly. Details of the model are given in Supplementary Table . g RIP3 higher-order assembly’s impact on MLKL phosphorylation. h Statistical analysis of 10 4 random models showing the influence of RIP3 assembly on MLKL phosphorylation efficiency, with approximately 3:1 ratio of RIP3 to RIP1 consistently yielding the highest efficiency at 4 h and 6 h. Box plots: centre line, median; box bounds, 25th and 75th percentiles (IQR); whiskers extend to the lowest and highest data points within 1.5×IQR from the lower and upper quartiles. To visualize the distribution, all simulation outcomes are overlaid as jittered dots. The overlaid solid line connects the per-condition mean of the 500 simulations to show the trend. For each condition, n = 500 independent stochastic simulations. i Three-color STORM of necrosomes in HA-RIP1/Flag-RIP3–expressing RIP1- KO HeLa cells treated with TSZ for 4 h, labeled for HA-RIP1 (green), Flag-RIP3 (purple), and p-MLKL (yellow). Insets highlight necrosome morphology with calculated Flag:HA ratios and p-MLKL localizations. Images are representative of two independent experiments. j Quantification of RIP3:RIP1 stoichiometry and relative p-MLKL levels (localizations) in individual necrosomes ( n = 122). Scale bars: 10 μm ( i , overview), 5 µm ( e , overview), 300 nm ( i , insets), and 100 nm ( e , insets). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used throughout this report: anti-RIP1 (Cell Signaling, 3493, 1:150 for immunofluorescence (IF), 1:1,000 for western blotting (WB)), anti-phospho Ser166 RIP1 (Cell Signaling, 65746, 1:1,000 for WB), anti-RIP3 (Cell Signaling, 13526, 1:1,000 for WB), anti-phospho Ser227 RIP3 (Abcam, ab209384, 1:1,000 for WB), anti-MLKL (Abcam, ab184718, 1:200 for IF; 1:1,000 for WB), anti-phospho Ser358 MLKL (Abcam, ab187091, 1:1,000 for WB), anti-HA (Santa Cruz, sc-7392, 1:200 for IF, 1:1,000 for WB; Cell Signaling, 3724, 1:200 for IF, 1:1,000 for WB; ABclonal, AE008, 1:200 for IF), anti-Flag (Abmart, M20008L, 1:1000 for IF, 1:5,000 for WB; Biolegend, 637301, 1:200 for IF), anti-GAPDH (Proteintech, 60004-1-Ig, 1:5,000 for WB), goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to Alexa Fluor 488, 568 or 647 (Thermo Fisher Scientific, A11034, A11004 or A21247, 1:1,000 for IF) and goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to CF 488 A, CF568 or CF 647 (Biotium, 20015, 20800, 20801, 20808 or 20809, 1:500 for IF).

Techniques: Transmission Assay, Expressing, Labeling, Phospho-proteomics

Journal: Nature Communications

Article Title: Decoding necrosome assembly: harmonizing signal amplification and attenuation through optimal RIP3 stoichiometry

doi: 10.1038/s41467-025-67098-5

Figure Lengend Snippet: Theoretical predictions illustrating the regulation of TNF ( a ), RIP3 ( b ), and MLKL ( c ) on RIP1-RIP3 assembly. Here, p-MLKL/MLKL tot represents system efficiency with MLKL tot indicating MLKL expression level. Maximum efficiency corresponds the optimal RIP3 assembly degrees under given conditions. Experimental validation of TNF ( d ), RIP3 ( f ), and MLKL ( h ) effects on RIP3 assembly via immunoblotting and STORM. Changes in RIP3 and MLKL phosphorylation, and corresponding rod-shaped RIP3 structures (STORM), are shown under varying TNF stimulation ( d ), doxycycline-induced RIP3 expression ( f ), and shRNA-mediated MLKL knockdown ( h ). e , g , i , Statistical analysis of areas and counts of rod-shaped RIP3 structures in STORM images from cells under different TNF doses ( e ), RIP3 expression levels ( g ), and MLKL expression levels ( i ). Experiments in ( d-i ) used Flag-RIP3-expressing HeLa cells treated with TSZ under graded TNF concentrations ( d,e ; n = 28,48,64,80 structures), induced RIP3 expression ( f , g ; n = 24,30,42,86), or MLKL knockdown ( h, i ; n = 40,64,88,94). RIP3/MLKL levels were quantified by immunoblot densitometry. Necrosome counts were presented as mean ± SD from three cells. j Co-immunoprecipitation in 293 T cells transiently expressing HA-RIP3, Flag-RIP3, and Myc-MLKL. Lysates were immunoprecipitated with anti-Flag beads, and inputs/IPs were immunoblotted for Flag, HA, and MLKL. HA-RIP3 binding to Flag-RIP3 was quantified as IP/input ratio. Data are presented as mean ± SD from three independent experiments. k–m Flag-RIP3–expressing wild-type or MLKL-KO HeLa cells treated with TSZ over time. Cell death was measured by PI uptake and shown as mean ± SD from three biological replicates ( k ). Dual-color STORM of RIP3 and p-MLKL ( l ); insets show necrosome morphology. RIP3 fibril lengths were quantified ( m ; n = 67/111 structures at 2 h, 64/135 at 4 h for WT/KO). Data in ( d , f , h and l ) are representative of two independent experiments. p values were calculated using unpaired two tailed t test ( j ), or Two-Way ANOVA Tukey’s multiple comparisons test ( k and m ). Scale bars: 10 μm (overviews in d , f , h , and l ) and 200 nm (insets in d , f , h , and l ). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used throughout this report: anti-RIP1 (Cell Signaling, 3493, 1:150 for immunofluorescence (IF), 1:1,000 for western blotting (WB)), anti-phospho Ser166 RIP1 (Cell Signaling, 65746, 1:1,000 for WB), anti-RIP3 (Cell Signaling, 13526, 1:1,000 for WB), anti-phospho Ser227 RIP3 (Abcam, ab209384, 1:1,000 for WB), anti-MLKL (Abcam, ab184718, 1:200 for IF; 1:1,000 for WB), anti-phospho Ser358 MLKL (Abcam, ab187091, 1:1,000 for WB), anti-HA (Santa Cruz, sc-7392, 1:200 for IF, 1:1,000 for WB; Cell Signaling, 3724, 1:200 for IF, 1:1,000 for WB; ABclonal, AE008, 1:200 for IF), anti-Flag (Abmart, M20008L, 1:1000 for IF, 1:5,000 for WB; Biolegend, 637301, 1:200 for IF), anti-GAPDH (Proteintech, 60004-1-Ig, 1:5,000 for WB), goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to Alexa Fluor 488, 568 or 647 (Thermo Fisher Scientific, A11034, A11004 or A21247, 1:1,000 for IF) and goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to CF 488 A, CF568 or CF 647 (Biotium, 20015, 20800, 20801, 20808 or 20809, 1:500 for IF).

Techniques: Expressing, Biomarker Discovery, Western Blot, Phospho-proteomics, shRNA, Knockdown, Immunoprecipitation, Binding Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Decoding necrosome assembly: harmonizing signal amplification and attenuation through optimal RIP3 stoichiometry

doi: 10.1038/s41467-025-67098-5

Figure Lengend Snippet: a Schematic workflow outlining the investigation of regulatory strategies using randomized signaling models. A TNF signaling network model was constructed (Supplementary Table ) and subjected to LHS method to sample parameter variables within physiological ranges, including stochastic stoichiometries of RIP3 assembly. Kinetic analysis was performed on all sampled models, with the top 5% identified based on MLKL phosphorylation efficiencies. Statistical analysis of these models was conducted to explore trends in RIP3 assembly under varying TNF, RIP3, and MLKL conditions. b Distribution of RIP3 ratios to RIP1 and corresponding MLKL phosphorylation efficiencies in the top 5% efficient models under different conditions. c Statistical results corresponding to the distribution of the three scenarios shown in panel b . Box plots: centre line, median; box bounds, 25th and 75th percentiles (IQR); whiskers extend to the lowest and highest data points within 1.5×IQR from the lower and upper quartiles. To visualize the distribution, all simulation outcomes are overlaid as jittered dots. The overlaid solid line connects the per-condition mean of the 500 simulations to show the trend. For each condition, n = 500 independent stochastic simulations; concentrations as indicated (nM). d Regulation of TNF, RIP3, and MLKL on RIP3 ratios. e , f Theoretical predictions and experimental validations of the impacts of TNF, RIP3, and MLKL on MLKL/RIP3 phosphorylation efficiencies. g Schematic illustrating strategic balance between necrosome abundance and RIP3 stoichiometries in cells: high necrosome abundance favors smaller RIP3 assemblies, while low abundance necessitates larger RIP3 assemblies. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used throughout this report: anti-RIP1 (Cell Signaling, 3493, 1:150 for immunofluorescence (IF), 1:1,000 for western blotting (WB)), anti-phospho Ser166 RIP1 (Cell Signaling, 65746, 1:1,000 for WB), anti-RIP3 (Cell Signaling, 13526, 1:1,000 for WB), anti-phospho Ser227 RIP3 (Abcam, ab209384, 1:1,000 for WB), anti-MLKL (Abcam, ab184718, 1:200 for IF; 1:1,000 for WB), anti-phospho Ser358 MLKL (Abcam, ab187091, 1:1,000 for WB), anti-HA (Santa Cruz, sc-7392, 1:200 for IF, 1:1,000 for WB; Cell Signaling, 3724, 1:200 for IF, 1:1,000 for WB; ABclonal, AE008, 1:200 for IF), anti-Flag (Abmart, M20008L, 1:1000 for IF, 1:5,000 for WB; Biolegend, 637301, 1:200 for IF), anti-GAPDH (Proteintech, 60004-1-Ig, 1:5,000 for WB), goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to Alexa Fluor 488, 568 or 647 (Thermo Fisher Scientific, A11034, A11004 or A21247, 1:1,000 for IF) and goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to CF 488 A, CF568 or CF 647 (Biotium, 20015, 20800, 20801, 20808 or 20809, 1:500 for IF).

Techniques: Construct, Phospho-proteomics

Journal: Nature Communications

Article Title: Decoding necrosome assembly: harmonizing signal amplification and attenuation through optimal RIP3 stoichiometry

doi: 10.1038/s41467-025-67098-5

Figure Lengend Snippet: a Schematic representation of the TNF-mediated assembly model for RIP1-FADD-caspase-8 complex in HeLa cells, validated by experimental caspase-8 activation data. Western blot analysis was performed for the indicated proteins, with the levels of cleaved caspase-8 quantified by relative band intensities. Data are representative of two independent experiments. b Model predictions illustrating the effect of reduced RIP1 expression on caspase-8 recruitment into RIP1 complex (upper panel), alongside corresponding experimental data using literature data (lower panel). Specifically, Caspase-8 levels in complex were quantified by western blotting and RIP1 levels were controlled by shRNA-mediated knockdown. c Diagram depicting the biphasic response driven by threshold and linear response behaviors of RIP3 and caspase-8 within necrosomes. d Modeling prediction of a biphasic response induced by decreasing RIP1 levels (upper panel), with experimental validation using literature data (lower panel). Specifically, p-RIP3 levels were quantified by western blotting using a phospho-specific RIP3 antibody and RIP1 levels were controlled by shRNA-mediated knockdown. e–g WT or RIP1-deficient HeLa cells treated with TNF plus cycloheximide (TC) for indicated durations. Cell viability was assessed using a CCK-8 assay and shown as mean ± SD from three biological replicates per group ( e ). Western blot analysis was performed for the indicated proteins, with the levels of c-FLIP and cleaved caspase-8 quantified by relative band intensities. Blots are representative of four independent experiments ( f ). Confocal images showing caspase-8 distribution in TC-treated cells. Insets highlight enlarged caspase-8 structures. Images are representative of two independent experiments ( g ). p values were calculated using Two-Way ANOVA Tukey’s multiple comparisons test. Scale bars, 30 μm (overviews) and 10 µm (insets). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used throughout this report: anti-RIP1 (Cell Signaling, 3493, 1:150 for immunofluorescence (IF), 1:1,000 for western blotting (WB)), anti-phospho Ser166 RIP1 (Cell Signaling, 65746, 1:1,000 for WB), anti-RIP3 (Cell Signaling, 13526, 1:1,000 for WB), anti-phospho Ser227 RIP3 (Abcam, ab209384, 1:1,000 for WB), anti-MLKL (Abcam, ab184718, 1:200 for IF; 1:1,000 for WB), anti-phospho Ser358 MLKL (Abcam, ab187091, 1:1,000 for WB), anti-HA (Santa Cruz, sc-7392, 1:200 for IF, 1:1,000 for WB; Cell Signaling, 3724, 1:200 for IF, 1:1,000 for WB; ABclonal, AE008, 1:200 for IF), anti-Flag (Abmart, M20008L, 1:1000 for IF, 1:5,000 for WB; Biolegend, 637301, 1:200 for IF), anti-GAPDH (Proteintech, 60004-1-Ig, 1:5,000 for WB), goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to Alexa Fluor 488, 568 or 647 (Thermo Fisher Scientific, A11034, A11004 or A21247, 1:1,000 for IF) and goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to CF 488 A, CF568 or CF 647 (Biotium, 20015, 20800, 20801, 20808 or 20809, 1:500 for IF).

Techniques: Activation Assay, Western Blot, Expressing, shRNA, Knockdown, Biomarker Discovery, CCK-8 Assay

Journal: Nature Communications

Article Title: Decoding necrosome assembly: harmonizing signal amplification and attenuation through optimal RIP3 stoichiometry

doi: 10.1038/s41467-025-67098-5

Figure Lengend Snippet: When cells are stimulated with TNF under specific conditions (e.g., cIAP1/2 depletion or IKK/TBK1 inhibition), RIP1 dissociates from TNFR1 and assembles into cytosolic supramolecular complexes containing RIP3, MLKL, FADD, and caspase-8 (middle schematic). Within these complexes, RIP3 adopts a rod-shaped configuration of approximately three molecules per RIP1, functioning as a signaling amplifier that confers an ultrasensitive response to RIP1 levels. RIP1 and caspase-8 assemble at an approximate 1:1 ratio, generating a linear activation response, while mutual inhibition between RIP3 and caspase-8 produces biphasic regulation of necroptotic signaling by RIP1. A RIP3:RIP1 ratio of roughly 3:1 optimizes MLKL phosphorylation efficiency. In the absence or inhibition of caspase-8 (left schematic), RIP3 stoichiometry becomes dynamically regulated: larger assemblies correlate with stronger signal amplification and more pronounced threshold responses. RIP1 and MLKL act as negative regulators of RIP3 homo-assembly, whereas RIP3 promotes its own polymerization. Efficient signal transmission requires a balance between the size of individual rod-like RIP3 assemblies and the total number of necrosomes, as larger complexes form in smaller numbers. When caspase-8 is present (right schematic), RIP1-recruited caspase-8 remains non-filamentous in the presence of high c-FLIP levels, leading to weak apoptotic signaling. Upon c-FLIP depletion, caspase-8 polymerizes into helical filaments that drive robust apoptosis. Although the precise optimal RIP1:RIP3 ratio may vary across systems and contexts, our large-scale stochastic simulations (Fig. ) recapitulate the deterministic model, indicating that these behaviors reflect conserved principles of supramolecular assembly.

Article Snippet: The following antibodies were used throughout this report: anti-RIP1 (Cell Signaling, 3493, 1:150 for immunofluorescence (IF), 1:1,000 for western blotting (WB)), anti-phospho Ser166 RIP1 (Cell Signaling, 65746, 1:1,000 for WB), anti-RIP3 (Cell Signaling, 13526, 1:1,000 for WB), anti-phospho Ser227 RIP3 (Abcam, ab209384, 1:1,000 for WB), anti-MLKL (Abcam, ab184718, 1:200 for IF; 1:1,000 for WB), anti-phospho Ser358 MLKL (Abcam, ab187091, 1:1,000 for WB), anti-HA (Santa Cruz, sc-7392, 1:200 for IF, 1:1,000 for WB; Cell Signaling, 3724, 1:200 for IF, 1:1,000 for WB; ABclonal, AE008, 1:200 for IF), anti-Flag (Abmart, M20008L, 1:1000 for IF, 1:5,000 for WB; Biolegend, 637301, 1:200 for IF), anti-GAPDH (Proteintech, 60004-1-Ig, 1:5,000 for WB), goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to Alexa Fluor 488, 568 or 647 (Thermo Fisher Scientific, A11034, A11004 or A21247, 1:1,000 for IF) and goat anti-rabbit, anti-mouse and anti-rat secondary antibodies conjugated to CF 488 A, CF568 or CF 647 (Biotium, 20015, 20800, 20801, 20808 or 20809, 1:500 for IF).

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Amplification, Transmission Assay