Necrostatin 1

Manufactured by Selleck Chemicals

Sourced in United States, China

Necrostatin-1 is a chemical compound that functions as a selective inhibitor of necroptosis, a form of programmed cell death. It acts by blocking the receptor-interacting protein kinase 1 (RIPK1), a key regulator of the necroptosis pathway. Necrostatin-1 has been widely used in research to study the mechanisms and implications of necroptosis in various biological systems.

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Necrostatin-1 (Nec-1) Necrostatin

63 protocols using necrostatin 1

Investigating Necroptosis Signaling Pathways

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The following commercial antibodies and reagents were used: Necrostatin-1 (S8037, Selleck); Necrostatin 1S (S8641, Selleck); Z-VAD-FMK (S0723, Selleck); TPCA-1(S2824, Selleck); PH-797804 (S2726, Selleck); SP600125 (S1460, Selleck); C-176 (S6575, Selleck); QNZ (S4902, Selleck); RIPK1 (1:1000, 551041, BD Biosciences); RIPK3 (1:1000, NBP1-77299, NOVUS); MLKL (1:2000, AP14272B, Abcepta); pMLKL (1:1000, 37333S, Cell Signaling Technology); Flag (1:1000, 20543-1-AP, Proteintech); caspase3 (1:1000, 19677-1-AP, Proteintech); HMGB1 (1:1000, 10829-1-AP, Proteintech); p65 (1:1000, 66535-1-Ig, Proteintech); GAPDH (1:10,000, 60004-1-Ig, Proteintech); Histone H3 (1:1000, 17168-1-AP, Proteintech);IL6 (1:1000, 66146-1-Ig, Proteintech); CD68 (1:1000, 28058-1-AP, Proteintech); TMEM119 (1:1000, 27585-1-AP, Proteintech); p-p65 (1:200, sc-136548, Santa Cruz); IKK (1:500, AF6009, Affinity); p-IKK (1:500, AF3013, Affinity); IKbα (1:1000, 10268-1-AP, Proteintech); p-IKbα (1:500, AF2002, Affinity); Ccl5 (1:500, AF5151, Affinity); IFNb1 (1:500, DF6471, Affinity); pTau396 (1:1000, 829,001, Biolegend); AT8 (1:1000, MN1020, Thermo); Goat Anti-Mouse-HRP IgG (1:10,000, 115-035-003, Jackson ImmunoResearch); Goat Anti-Rabbit-TRITC IgG (1:200, 111-025-045, Jackson ImmunoResearch); Goat Anti-Rabbit-HRP IgG (1:10,000, 111-005-003, Jackson ImmunoResearch).

Dong Y., Yu H., Li X., Bian K., Zheng Y., Dai M., Feng X., Sun Y., He Y., Yu B., Zhang H., Wu J., Yu X., Wu H, & Kong W. (2022). Hyperphosphorylated tau mediates neuronal death by inducing necroptosis and inflammation in Alzheimer’s disease. Journal of Neuroinflammation, 19, 205.

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AML Cell Lines and Patient Samples

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HL60, MOLM14, SET2, MV4-11, OCI-AML2, OCI-AML3, K562, THP1, UT7-EPO, SKM1, NB4 and KASUMI AML cell lines were used. Patients provided written informed consent in accordance with the Declaration of Helsinki. Bone marrow (BM) samples were obtained from five patients with newly diagnosed AML (characteristics provided in the Online Supplementary Table S1). Cells were cultured in RPMI with glutamine (Gibco61870, Life Technologies® Saint Aubin, France) supplemented with 10% fetal bovine serum (FBS) and 4 mM glutamine. All AML cell lines were certified using their microsatellite identity (characteristics provided in Online Supplementary Table S2). Ferrostatin-1, necrostatin-1, necrostatin-1S, necrosulfonamide, QVD-OPH, APR-246 for the in vitro study, erastin and RSL3 were sourced from Selleckchem (Houston, TX). Chloroquine and doxycycline were obtained from Sigma–Aldrich (Saint-Louis, MO). FINO2 was purchased from Cayman Chemicals (Ann Arbor, MI). The APR-246 reagent used in the in vivo study was provided by APREA therapeutics (Solna, Sweden).

Birsen R., Larrue C., Decroocq J., Johnson N., Guiraud N., Gotanegre M., Cantero-Aguilar L., Grignano E., Huynh T., Fontenay M., Kosmider O., Mayeux P., Chapuis N., Sarry J.E., Tamburini J, & Bouscary D. (2021). APR-246 induces early cell death by ferroptosis in acute myeloid leukemia. Haematologica, 107(2), 403-416.

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Cell Death Inhibitor Assay

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Drugs or cell death inhibitors are used at the following concentrations: Necrostatin-1s (10 μg/mL, Selleck Chemicals), Ferrostatin-1 (2 μM, XcessBio), Liproxstatin-1 (2 μM, Sellek Chemicals), Vitamin E (125 μM, Sigma Aldrich), Cyclosporin A (1 μM, Sigma Aldrich), ZVAD-FMK (20 μM, Abcam), NIM-811 (1 μM, MedChem Express), FK506 (1 μM, ApexBio), Nutlin-3a (10 μM, ApexBio). Cell death inhibitors were added to cells at time of plating for the duration of the experiment.

Acosta J., Li Q., Freeburg N.F., Murali N., Indeglia A., Grothusen G.P., Cicchini M., Mai H., Gladstein A.C., Adler K.M., Doerig K.R., Li J., Ruiz-Torres M., Manning K.L., Stanger B.Z., Busino L., Murphy M., Wan L, & Feldser D.M. (2023). p53 restoration in small cell lung cancer identifies a latent cyclophilin-dependent necrosis mechanism. Nature Communications, 14, 4403.

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Investigating Cell Line Responses to Small Molecules

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Huh7 and SK-HEP-1 cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea). Dulbecco’s modified Eagle medium, RPMI-1640 medium, fetal bovine serum, L-glutamine, penicillin, and streptomycin were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Sulfasalazine, ferrostatin-1, necrostatin-1, deferoxamine, and 1-methyl-D-tryptophan were purchased from Merck KGaA (Darmstadt, Germany). Erastin, RSL3, necrostatin-1s, and GSK2982772 and Z-VAD-FMK were obtained from Selleckchem (Houston, TX, USA). TNFα was supplied from PeproTech (Cranbury, NJ, USA) and SM164 was obtained from AdooQ Bioscience (Irvine, CA, USA).

Yuk H., Abdullah M., Kim D.H., Lee H, & Lee S.J. (2021). Necrostatin-1 Prevents Ferroptosis in a RIPK1- and IDO-Independent Manner in Hepatocellular Carcinoma. Antioxidants, 10(9), 1347.

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Cell Viability Assay Protocol

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Cell viability was measured as described previously using the Cell Counting Kit-8 (CCK8) (Enogene, Nanjing, China). In brief, cells were seeded in 96-well plates at a density 4 × 10³–8 × 10³ cells per well and incubated for 24 h. Then, the cells were exposed to different treatment. Cells were treated with glucose (Solarbio, Beijing, China)., ferroptosis inducers, erastin (Selleckchem, Houston, USA), or RSL3 (Selleckchem, Houston, USA); Liraglutide; cell death inhibitors, including ferrostatin-1 (Selleckchem, Houston, USA), necrostatin-1 s (Selleckchem, Houston, USA), or Z-VAD-fmk (Selleckchem, Houston, USA); AMPK inhibitor, compound C (MedChemExpress, USA); After that, the medium in each well was replaced with 100 µl fresh medium containing 10 µl CCK8 reagent. After incubation for 1 h at 37 °C, 5% CO₂ incubator. The absorbance at a wavelength of 450 nm was measured using a Multiskan Spectrum (Thermo Scientific, Waltham, MA, USA).

Guo T., Yan W., Cui X., Liu N., Wei X., Sun Y., Fan K., Liu J., Zhu Y., Wang Z., Zhang Y, & Chen L. (2023). Liraglutide attenuates type 2 diabetes mellitus-associated non-alcoholic fatty liver disease by activating AMPK/ACC signaling and inhibiting ferroptosis. Molecular Medicine, 29, 132.

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High-Throughput Oligodendrocyte Viability Assay

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OPCs were seeded in 384-well plates (PerkinElmer, 6057500) pre-coated with poly-D-lysine and laminin (Sigma, L2020) at a density of 12,500 cell per well and allowed to attach for 1 hour at 37°C. Cell death inhibitors quinoline-Val-Asp-Difluorophenoxymethylketone (QVD-OPH) (Selleck, S7311), ferrostatin-1 (Selleck, S7243), and necrostatin-1 (Selleck, S8037), were added using a Janus automated workstation and 50 nL solid pin tool attachment in 8-point dose response (80 nM to 10 µM), and incubated for 1 hour at 37°C. Methyltrioctylammonium chloride or tributyltetradecylphosphonium chloride was added to all wells at IC₉₀ concentrations (approximately 100 nM), and oligodendrocytes were allowed to develop for 72 hours. Negative control wells contained only methyltrioctylammonium chloride or tributyltetradecylphosphonium chloride. Positive control wells contained vehicle (DMSO). Cells were fixed with 4% Paraformaldehyde (Electron Microscopy Sciences, HP1–100Kit) and stained with and DAPI (1 μg/mL, Sigma, D8417). Imaging was performed with the Operetta High Content Imaging and Analysis system (PerkinElmer) and the PerkinElmer Harmony and Columbus software was used to quantify DAPI-positive nuclei.

Cohn E.F., Clayton B.L., Madhavan M., Yacoub S., Federov Y., Paul-Friedman K., Shafer T.J, & Tesar P.J. (2023). Pervasive environmental chemicals impair oligodendrocyte development. bioRxiv.

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PFOA-Induced Cellular Damage in ALC Cells

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The mouse ameloblast-lineage cells (ALC cells), which are an immortalized cell line from the mouse mandibular molar (Nakata et al., 2003 ) were used in this study. ALC cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10 % fetal bovine serum. Indicated concentrations of PFOA (Sigma-Aldrich, St. Louis, MO, USA) were diluted with cell culture medium. All inhibitors used in this study were added 1 h prior to PFOA treatment. N-acetyl cysteine (NAC) (Thermo Fisher Scientific), which inhibits reactive oxygen species (ROS) production, was diluted with cell culture medium. The necroptosis inhibitors, RIP1 (Necrostatin-1) and PD98059 (ERK inhibitor) (Selleck Chemicals, Houston, TX, USA) were diluted with dimethyl sulfoxide (DMSO). A final concentration of 0.04 % DMSO (vehicle) served as a control and was equivalent to the amount of DMSO in each sample.

Fujiwara N., Yamashita S., Okamoto M., Cooley M.A., Ozaki K., Everett E.T, & Suzuki M. (2023). Perfluorooctanoic acid-induced cell death via the dual roles of ROS-MAPK/ERK signaling in ameloblast-lineage cells. Ecotoxicology and environmental safety, 260, 115089.

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Quantifying Cell Cytotoxicity via LDH Assay

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ESCs were seeded at a density of 2 × 10⁵ cells/mL in the indicated media type in a clear 96-well plate. Inhibitors were administered 24 hr after seeding at a concentration of 50 μM (Z-VAD-FMK, ApexBio; Necrostatin-1, Selleckchem). At the indicated timepoints, lactate dehydrogenase (LDH) activity was quantified in the supernatant using the Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific) according to the manufacturer’s instructions. A_680nm values were first subtracted as background noise. Then, absorbance from an average of 3 media-only wells (reflecting background LDH activity) was subtracted from every sample’s A_490nm value. Data were normalized to wells that had been lysed completely using the provided lysis buffer to establish a benchmark for 100% cell death.

LeBlanc L., Lee B.K., Yu A.C., Kim M., Kambhampati A.V., Dupont S.M., Seruggia D., Ryu B.U., Orkin S.H, & Kim J. (2018). Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation. eLife, 7, e40167.

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Comprehensive Cell Signaling Reagents Protocol

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Dulbecco's modified Eagle's medium (DMEM) and foetal bovine serum (FBS) were obtained from Gibco. Earle's balanced salt solution (EBSS) and propranolol were purchased from Sigma‐Aldrich. Rapamycin (RAPA), 3‐methyladenine (3‐MA) and chloroquine (CQ) were obtained from MedChemExpress, and ZVAD‐FMK, necrostatin‐1, liproxstatin‐1, SB203580, SP600125, SC79 and 740Y‐P were purchased from SelleckChem. Then, these reagents were dissolved in dimethyl sulfoxide (DMSO) (MP Biomedicals or water and stored at −80°C. Primary antibodies against Akt, p‐Akt, p38 MAPK, p‐p38 MAPK, JNK, p‐JNK, Erk1/2 and p‐Erk1/2 were purchased from Cell Signalling Technology. Primary antibodies against alpha‐smooth muscle actin (α‐SMA), fibronectin (FN), LC3B, P62, p‐PI3K p85, PI3K p85, ATG9b, ATG9a, mTOR, p‐mTOR, ATG12, ATG5 and anti‐ubiquitin were procured from Abcam. Primary antibodies against beta‐actin, alpha‐tubulin, GAPDH and HRP‐conjugated secondary antibodies were obtained from Proteintech. DyLight 549‐conjugated and DyLight 488‐conjugated secondary antibodies were provided by Abbkine.

Wang S., Ding Q., Xiu A., Xia Y., Wang G, & Zhang C. (2023). Upregulation of ATG9b by propranolol promotes autophagic cell death of hepatic stellate cells to improve liver fibrosis. Journal of Cellular and Molecular Medicine, 28(2), e18047.

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Cell Viability Assay with Small Molecules

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For cell viability experiments, cells were treated with a range of concentrations of the indicated small molecule compounds, and relative viability was measured by the CellTiter-Glo Assay. Cells were seeded in 96-well black-wall tissue culture-treated plates at 2,000 to 3,000 cells per well (or 10,500 for primary human monocytes and macrophages), to reach 30% confluency the following day. Cells were treated with compounds 18–24 h after seeding using an HP D300e Digital Dispenser with three biological replicates per condition. For BSA-palmitate and BSA-control experiments, compounds were added manually to cells. 66–72 h after compound treatment, viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) as per the manufacturer’s instructions. Unless otherwise specified, relative viability was normalized to the untreated condition. Regression fit curves were computed in Prism 8 or 9 (GraphPad) using four-parameter inhibition nonlinear regression. The mean and standard deviation for three biological replicates of each data point were calculated. The following compounds were used: RSL3 (Selleck Chem), ML210 (Sigma Aldrich), erastin (Selleck Chem), FIN56 (Selleck Chem), Ferrostatin-1 (Sigma Aldrich), Z-VAD-FMK (Selleck Chem), Necrostatin-1 (Selleck Chem), BSA-palmitate (Cayman Chem), BSA-control (Cayman Chem).

Mahr K., Hillen W, & Titgemeyer F. (2000). Carbon Catabolite Repression in Lactobacillus pentosus: Analysis of the ccpA Region. Applied and Environmental Microbiology, 66(1), 277-283.

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