Necrosulfonamide nsa

Manufactured by Merck Group

Sourced in Germany

Necrosulfonamide (NSA) is a laboratory reagent used in cell biology research. It functions as a specific inhibitor of the enzyme Mixed Lineage Kinase Domain-Like Protein (MLKL), which is involved in the process of necroptosis, a form of programmed cell death. NSA is utilized by researchers to study the mechanisms and signaling pathways associated with necroptosis in various cellular systems.

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10 protocols using necrosulfonamide nsa

Cytotoxicity Assays in Cell Cultures

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Organic solvents for preparing the extract, such as dichloromethane, methanol, and DMSO, labels such as Thiazolyl Blue Tetrazolium Bromide (MTT), Crystal Violet, Hoechst 33342, and 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA), and N-acetyl-L-cysteine (NAC), 4-phenylbutyric acid (4-PBA), chloroquine (Chlo), necrostatin-1 (Nec-1), and necrosulfonamide (Nsa) compounds were purchased from Sigma-Aldrich (Europe). Z-VAD-FMK (zVAD) was obtained from APExBIO Technology, United States.

Ruiz-Torres V., Forsythe N., Pérez-Sánchez A., Van Schaeybroeck S., Barrajón-Catalán E, & Micol V. (2021). A Nudibranch Marine Extract Selectively Chemosensitizes Colorectal Cancer Cells by Inducing ROS-Mediated Endoplasmic Reticulum Stress. Frontiers in Pharmacology, 12, 625946.

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Pharmacological Inhibition of Cell Death

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GGTI2133 was bought from Santa Cruz Biotechnology (Dallas, USA, #1217480-14-2). FTI was from Selleck (Texas, USA, #S7465). CsA was from Selleck (#S2286). NAC was from Sigma (#A7250). Necrosulfonamide (NSA, # 432531) and Necrostatin-1 (Nec-1) (Sigma #480065) were from Sigma. Nec-1s (VWR, Pennsylvania, USA, #852391-15-2), zvad-fmk (Sigma #V116), CT04 (Cytoskeleton, Denver, USA, #CT04), Ehop016 (Selleck #S7319). During the experiments, cells were pretreated with inhibitors for 30 minutes and then went for following stimulations.

Chen J., Zhang X., Li L., Ma X., Yang C., Liu Z., Li C., Fernandez-Cabezudo M.J., al-Ramadi B.K., Wu C., Huang W., Zhang Y., Zhang Y, & Liu W. (2021). Farnesyl pyrophosphate is a new danger signal inducing acute cell death. PLoS Biology, 19(4), e3001134.

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Quantifying MLKL Expression in PBMCs and Fibroblasts

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Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Primary fibroblasts were obtained from skin biopsies taken from the patients and their sister at Aarhus University Hospital, Denmark and were processed in accordance with StemBANCC standard operating procedures (University of Oxford). As an additional control, primary fibroblasts (‘F1’) were purchased from the ATCC (PCS-201-012). Fibroblasts were stimulated with human interferon (IFN)-γ (PeproTech) for MLKL upregulation, and were also cultured with 25 μM of the protease inhibitor MG-132 (Sigma-Aldrich) or 2 μM of the MLKL inhibitor necrosulfonamide (NSA) (Millipore) for 24 h. Real-time quantitative PCR was performed using TaqMan® Gene Expression Assays MLKL_Hs00930421_m1 and GAPDH_Hs02758991_g1. Real-time quantitative PCR was performed using TaqMan® Gene Expression Assays MLKL_Hs00930421_m1 and GAPDH_Hs02758991_g1. Expression of MLKL mRNA in PBMCs was assessed in triplicate. Relative transcript levels are expressed as 2^−∆Ct, where ∆Ct = (MLKL cycle threshold)−(GAPDH cycle threshold). MLKL mRNA expression in IFNγ-stimulated fibroblasts was measured in three independent experiments. Protein-level expression was assessed by Western blotting.

Faergeman S.L., Evans H., Attfield K.E., Desel C., Kuttikkatte S.B., Sommerlund M., Jensen L.T., Frokiaer J., Friese M.A., Matthews P.M., Luchtenborg C., Brügger B., Oturai A.B., Dendrou C.A, & Fugger L. (2020). A novel neurodegenerative spectrum disorder in patients with MLKL deficiency. Cell Death & Disease, 11(5), 303.

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Investigating Cell Signaling Pathways

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Diphenyleneiodonium (DPI), apocynin (APO), phorbol 12-myristate 13-acetate (PMA), LY294002, carboxymethylcellulose sodium salt and dextran from Leuconostoc spp. were purchased from Sigma-Aldrich. Hoechst 33342, rabbit anti-Mouse IgG secondary antibody, HRP, goat anti-Mouse IgG1 secondary antibody, PE, Qubit dsDNA HS assay kit and 3-(4,5-Dimethylthiazol-2-yl)−2,5-Diphenyltetrazolium Bromide (MTT) were from Invitrogen. PD98059, SB203580 and Cl-Amidine were from Cayman Chemical. Ham’s F-12 nutrient mix, Opti-MEM, DMEM, RPMI 1640 and fetal bovine serum (FBS) were from Gibco. Anti-neutrophil elastase antibody was from Abcam. Anti-RSV fusion protein antibody and necrosulfonamide (NSA) were from Millipore. Goat anti-rabbit IgG secondary antibody, Cy3 was from Chemicon International. Mouse anti-human myeloperoxidase, PE (MPO) was from BD Biosciences. The 7-Cl-O-Nec-1 (Nec-1s) and GW42X were a gift from Dr. Ricardo Weinlich (Hospital Israelita Albert Einstein, São Paulo, Brazil). Ficoll-Paque PLUS was from GE Healthcare. CytoTox 96 Non-Radioactive Cytotoxicity Assay was from Promega. Falcon 8-well culture slides were from Corning.

Muraro S.P., De Souza G.F., Gallo S.W., Da Silva B.K., De Oliveira S.D., Vinolo M.A., Saraiva E.M, & Porto B.N. (2018). Respiratory Syncytial Virus induces the classical ROS-dependent NETosis through PAD-4 and necroptosis pathways activation. Scientific Reports, 8, 14166.

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Scanning Electron Microscopy of Neutrophil Responses to Crystals

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Human neutrophils seeded in 8 well chamber slides were exposed to (200–500 µg/ml) crystals like calcium oxalate (Alfa Aesar, Karlsruhe, Germany), MSU (Invivogen, Toulouse, France), calcium phosphate (Chem Cruz, Heidelberg, Germany), cysteine (Sigma life sciences, Germany), cholesterol (Invivogen), crocidolite asbestos (SPI-CHEM, West Chester, PA), and silica (Alfa Aesar) and were incubated at 37 °C in 5% carbon dioxide atmosphere for 2 hours. For some experiments, neutrophils were pre-treated with 5 µM necrosulfonamide (NSA, Millipore, Schwalbach, Germany) for 30 minutes. Supernatants were removed and the samples were fixed in 3% glutaraldehyde in phosphate buffered saline (PBS), washed in 0.1 M Soerensen’s phosphate buffer, dehydrated in an ascending ethanol series (30–100%) and dried in hexamethyl disilazan (Sigma-Aldrich, Steinheim, Germany). The samples were analysed using a scanning electron microscope (ESEM XL 30 FEG, FEI, Eindhoven, Netherlands) by 0,8T with acceleration voltage of 10 kV. After the EDX analysis (EDAX Genisis System) they were sputtered with a 12.5 nm gold palladium layer and analyzed in a high vacuum environment.

Desai J., Foresto-Neto O., Honarpisheh M., Steiger S., Nakazawa D., Popper B., Buhl E.M., Boor P., Mulay S.R, & Anders H.J. (2017). Particles of different sizes and shapes induce neutrophil necroptosis followed by the release of neutrophil extracellular trap-like chromatin. Scientific Reports, 7, 15003.

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Endothelial Cell Culture and Stimulation

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Human umbilical vein endothelial cells (HUVEC), human umbilical artery endothelial cells (HUAEC), human dermal microvascular endothelial cells (HDMEC), Endothelial Cell Growth Medium (C-22010), and Endothelial Cell Growth Medium MV (C-22020) were purchased from Promocell. Human and mouse TNFα were purchased from R&D (human TNFα, 210-TA-100/CF; mouse TNFα, 410-MT-500/CF). Necrosulfonamide (NSA) was purchased from Millipore (480073). IKKα/β inhibitor TPCA-1 was obtained from Selleck (S2824). Nec-1s, SM164, and zVAD-fmk were provided by Prof. J. Yuan.

Dai J., Zhang C., Guo L., He H., Jiang K., Huang Y., Zhang X., Zhang H., Wei W., Zhang Y., Lu L, & Hu J. (2020). A necroptotic-independent function of MLKL in regulating endothelial cell adhesion molecule expression. Cell Death & Disease, 11(4), 282.

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Characterization of calcium signaling

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Ac-DEVD-CHO was purchased from Selleck Chemicals. ATP2B1 antibody (#ab3528) was purchased from Abcam. Necrosulfonamide (NSA) and SGMS1 antibodies (#ABC732) were purchased from Merck Millipore. β-actin antibody (#4970) was purchased from Cell Signaling Technology, Inc. α-hemolysin, BAPTA-AM, Chloroquine, 2-hydroxypropyl-β-cyclodextrin (HPβCD), EDTA, methyl-β-cyclodextrin (MβCD), sea nettle (Chrysaora quinquecirrha) venom (SN), sphingomyelinase and streptolysin O (SLO) were purchased from Sigma-Aldrich. Caloxin 1B3 (TIPKWISIIQALRGGGSK-amide) and 2A1 peptide (VSNSNWPSFPSSGGG-amide) was prepared by custom synthesis from Mimotopes.

Lau M.T., Manion J., Littleboy J.B., Oyston L., Khuong T.M., Wang Q.P., Nguyen D.T., Hesselson D., Seymour J.E, & Neely G.G. (2019). Molecular dissection of box jellyfish venom cytotoxicity highlights an effective venom antidote. Nature Communications, 10, 1655.

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Apoptosis and Necroptosis Inhibitor Assay

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Human Necrostatin-1 (Nec-1, Sigma), GSK872 (a gift from Anaxis Pty, Ltd.), coumermycin (Sigma), Necrosulfonamide (NSA, Merck Millipore), Propidium iodide (Sigma), Sytox Green (Thermo Fisher), puromycin (Thermo Fisher), N-ethylmaleimide (NEM, Sigma), deubiquitylase (DUBs) made in house (Hospenthal et al., 2015) , complete protease inhibitor cocktail (Roche), Bafilomycin A1 (BAF, Enzo), PS341 (Sigma).

Liu Z., Dagley L.F., Shield‐Artin K., Young S.N., Bankovacki A., Wang X.S., Tang M.J., Howitt J., Stafford C.A., Nachbur U., Fitzgibbon C., Garnish S.E., Webb A.I., Komander D., Murphy J.M., Hildebrand J.M., & Silke J. (2021). Oligomerization-driven MLKL ubiquitylation antagonises necroptosis.

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Poly(I:C)-Induced Cell Death Analysis

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Poly(I:C) HMW was purchased from InvivoGen (San Diego, California, USA). Smac mimetic (SM-164) was a gift from S. Wang (University of Michigan, Ann Arbor, Michigan, USA). Pan-caspase inhibitor (z-VAD-FMK), GSK’782, necrosulfonamide (NSA), Bay11–7082, U0126, SP600125 and SB203580 were purchased from Calbiochem (Merck Millipore, Darmstadt, Germany). Necrostatin-1 (Nec-1) were purchased from Sigma (St Louis, Missouri, USA). TNF-α was purchased from R&D systems (Minneapolis, Minnesota, USA). TLR3 inhibitor (CuCpt4a) was purchased from APExBIO (Boston, Massachusetts, USA). Antibodies for Western blot were purchased from commercial available providers as following: anti-RIPK1 (610459) was from BD Biosciences (San Jose, California, USA); anti-TLR3 (6961), anti-RIPK3 (8457), anti-cIAP1 (7065), anti-cIAP2 (3130), anti-caspase-8 (9746), anti-caspase-3 (9662), anti-PARP-1 (9542) and anti-actin (4970) were from Cell signaling (Danvers, Massachusetts, USA); anti-MLKL (ab184718) was from Abcam (Cambridge, UK).

Lomphithak T., Choksi S., Mutirangura A., Tohtong R., Tencomnao T., Usubuchi H., Unno M., Sasano H, & Jitkaew S. (2020). Receptor-interacting protein kinase 1 is a key mediator in TLR3 ligand and Smac mimetic-induced cell death and suppresses TLR3 ligand-promoted invasion in cholangiocarcinoma. Cell Communication and Signaling : CCS, 18, 161.

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Inhibition of Necroptosis in RSV-Infected AECs

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Human AECs from healthy paediatric (age 2-3 years) donors (ID 28195, 28563, 28385, and 29055) were obtained commercially (Lonza). Purified RSV A2 stocks were prepared as previously described (20). Non-differentiated (submerged) or air-liquid interface (ALI)differentiated AECs were grown and infected with RSV as previously described (21, 22) . One hour prior to RSV infection, the cells were pre-treated with a pRIPK1inhibitor (Necrostatin 1s; Nec-1s, 2.2 µM; Biovision) (23), which reduces RIPK1 phosphorylation, an MLKL inhibitor (necrosulfonamide; NSA; 10 µM; Merck) (24), which prevents MLKL translocation to the plasma membrane, a caspase-9 inhibitor (Z-LEHD-FMK; 2 µM; Biovision), and/or a caspase-8 inhibitor (Z-IETD-FMK; 2 µM; Biovision). After 12 or 24 hours, supernatant was collected and stored at -80°C, and the cells fixed with neutral buffered 10% formalin solution (Sigma) for 15 mins.

Simpson J., Loh Z., Ullah M.A., Lynch J.P., Werder R.B., Collinson N., Zhang V., Dondelinger Y., Bertrand M.J.M., Everard M.L., Blyth C.C., Hartel G., Van Oosterhout A.J., Gough P.J., Bertin J., Upham J.W., Spann K.M, & Phipps S. (2020). Respiratory Syncytial Virus Infection Promotes Necroptosis and HMGB1 Release by Airway Epithelial Cells. American journal of respiratory and critical care medicine, 201(11).

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