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Necrostatin 1

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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 activity of the receptor-interacting serine/threonine-protein kinase 1 (RIPK1), a key regulator of necroptosis. Necrostatin-1 is commonly used in research applications to study the mechanisms and effects of necroptosis.

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Lab products found in correlation

Ferrostatin 1, by Cayman Chemical (5 mentions) Dimethyl sulfoxide (dmso), by Merck Group (5 mentions) Z vad ome fmk, by Cayman Chemical (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Sb203580, by Selleck Chemicals (2 mentions) Chloroquine, by Merck Group (2 mentions) Z vad fmk, by Selleck Chemicals (2 mentions) Staurosporine, by Merck Group (2 mentions) Ac devd cho, by Selleck Chemicals (1 mentions) Ac devd cho, by Merck Group (1 mentions) Sp600125, by Selleck Chemicals (1 mentions) Bafilomycin a1, by Cayman Chemical (1 mentions) As1842856, by Selleck Chemicals (1 mentions) Pd0325901, by Selleck Chemicals (1 mentions) Trichloroacetic acid, by Merck Group (1 mentions) α tubulin dm1a, by Merck Group (1 mentions) Atf3 c19, by Santa Cruz Biotechnology (1 mentions) β actin ac 74, by Merck Group (1 mentions) Cleaved caspase 3 5a1e, by Cell Signaling Technology (1 mentions) Mcl 1 s 19, by Santa Cruz Biotechnology (1 mentions)

18 protocols using necrostatin 1

1

Inhibitors for Cell Signaling Assays

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Trichloroacetic acid was obtained from Sigma-Aldrich (St. Louis, MO, USA). Bafilomycin A1 (#11038), necrostatin-1 (#11658) and ferrostatin-1 (#17729) were obtained from Cayman chemical (Ann Arbor, MI, USA). PD0325901 (#S1036), SB203580 (#S1076), AS1842856 (#S8222), SP600125 (#S1460) and Ac-DEVD-CHO (#S7901) were purchased from Selleck (Plymouth, MI, USA). For preparing stocks for the inhibitors, Ac-DEVD-CHO was dissolved in ultrapure deionized water; the others were dissolved in DMSO purchased from Sigma-Aldrich (St. Louis, MO, USA). The final DMSO concentration in fish medium did not exceed 0.1%.
Ren J., Long Y., Liu R., Song G., Li Q, & Cui Z. (2021). Characterization of Biological Pathways Regulating Acute Cold Resistance of Zebrafish. International Journal of Molecular Sciences, 22(6), 3028.
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2

Antibody Acquisition and Small Molecule Procurement

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Antibodies were purchased as follows: BIM (C34C5), BCL-XL (54H6), CREB (48H2) and cleaved caspase-3 (5A1E) from Cell Signaling Technology (Danvers, MA, USA); NOXA (114C307.1) from Thermo Fisher Scientific (Waltham, MA, USA); ATF-3 (C-19) and MCL-1 (S-19) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); KLF4 (56CT5.1.6) from Bioss, Inc. (Woburn, MA, USA); α-tubulin (DM1A) and β-actin (AC-74) from Sigma-Aldrich (Tokyo, Japan). APTO-253 was purchased from MedChemExpress, LLC (Monmouth Junction, NJ, USA). Q-VD-OPh, Doxorubicin, etoposide, SP600125, SB203580, PD184352, and U0126 were purchased from Calbiochem (San Diego, CA, USA). Hydroxychloroquine (HCQ), Necrostatin-1 and Ferrostatin-1 were purchased from Cayman Chemical (Ann Arbor, MI, USA).
Nakajima W., Miyazaki K., Asano Y., Kubota S, & Tanaka N. (2021). Krüppel-Like Factor 4 and Its Activator APTO-253 Induce NOXA-Mediated, p53-Independent Apoptosis in Triple-Negative Breast Cancer Cells. Genes, 12(4), 539.
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3

Ferroptosis Inhibitors Protocol

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GAA was obtained from Selleck (Houston, TX, USA). Dimethyl sulfoxide (DMSO) and 1,10-Phenanthrolin (phen) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Era, RSL3, Fe-SP, Fer-1, Lip-1, and Necrostatin-1 (Nec-1) were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). ZVAD-FMK was obtained from BioVision (Milpitas, CA, USA).
Lin J.H., Yang K.T., Ting P.C., Luo Y.P., Lin D.J., Wang Y.S, & Chang J.C. (2021). Gossypol Acetic Acid Attenuates Cardiac Ischemia/Reperfusion Injury in Rats via an Antiferroptotic Mechanism. Biomolecules, 11(11), 1667.
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4

Chemical Compounds for Cell Death Study

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1S,3R-RSL3 (referred to as RSL3) (#19288), Ferrostatin-1 (#17729), idebenone (#15475), Deferoxamine (DFO, #14595), Erastin2 (#27087), ML162 (#20455), ZVAD(OMe)-FMK (#27421), Necrostatin-1 (#11658), IKE (Imidazole Ketone Erastin, #27088), Tocopherol (#25985) Auranofin(#15316), Sulfasalazine(#15025) were all purchased from Cayman Chemical. Etoposide (#E1383), chloroquine (#C6628), and sodium selenite (#S5261), were all purchased from Sigma-Aldrich. Blasticidin S HCl (A1113903), Puromycin (A1113803), Hygromycin B (#10687010), SYTOX Green Dead Cell Stain (#34860), and MitoTracker Deep Red FM (M22426) were all purchased from Thermo Fisher Scientific.
Li Z., Ferguson L., Deol K.K., Roberts M.A., Magtanong L., Hendricks J.M., Mousa G.A., Kilinc S., Schaefer K., Wells J.A., Bassik M.C., Goga A., Dixon S.J., Ingolia N.T, & Olzmann J.A. (2022). Ribosome stalling during selenoprotein translation exposes a ferroptosis vulnerability. Nature chemical biology, 18(7), 751-761.
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5

Apoptosis and Necroptosis Assays

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RPMI 1640 powder was purchased from Nissui Pharmaceutical (Tokyo, Japan), and l-glutamine was purchased from Wako Pure Chemical Industries (Osaka, Japan). Allophycocyanin-coupled (APC) Annexin V/7-Amino-actinomycin D (7-AAD) kit was purchased from BioLegend (San Diego, CA, USA). Annexin V- fluorescein isothiocyanate (FITC) was purchased from MBL (Nagoya, Japan), and Hoechst 33342 was purchased from Dojindo (Rockville, MD, USA). Necrostatin-1 and ferrostatin-1 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA), and WST-1 was purchased from Roche (Basel, Switzerland). Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) was purchased from Cell Signaling Technology (Danvers, MA, USA). All organic solvents were purchased from Kanto Chemicals (Tokyo, Japan).
Yun Y., Shioura M., Hitotsuyanagi Y., Yotsumoto S., Takahashi Y., Aoyagi Y., Kinoshita T., Takeya K, & Inoue H. (2021). Garcinielliptone G from Garcinia subelliptica Induces Apoptosis in Acute Leukemia Cells. Molecules, 26(9), 2422.
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6

Compound Acquisition and Stock Preparation

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Compounds were purchased as follows: decitabine from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); cisplatin, doxorubicin and Q-VD-OPh from Calbiochem (San Diego, CA, USA); paclitaxel from Sigma-Aldrich; and hydroxychloroquine and necrostatin-1 from Cayman Chemical (Ann Arbor, MI, USA). Stock solutions of decitabine were prepared in dimethyl sulfoxide (DMSO; Sigma-Aldrich) at concentrations of 0.5 and 1 mM.
Nakajima W., Miyazaki K., Sakaguchi M., Asano Y., Ishibashi M., Kurita T., Yamaguchi H., Takei H, & Tanaka N. (2022). Epigenetic Priming with Decitabine Augments the Therapeutic Effect of Cisplatin on Triple-Negative Breast Cancer Cells through Induction of Proapoptotic Factor NOXA. Cancers, 14(1), 248.
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7

Cell Culture and Antibody Reagents

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MDA-MB-231 cells were maintained in the media of DMEM/F12 with 10% FBS, and SUM159 cells were cultured in DMEM/F12 with 10% FBS containing hydrocoretisone (1 μg/ml). The following antibodies were purchased: cGAS (Arigo Biolaboratories); AMPKα, pT172-AMPKα, and pS172-TBK1 (Cell Signaling Technology); TBK1 (Novus Biologicals); β-actin (Santa Cruz Biotechnology), mouse and rabbit secondary antibody-conjugated horseradish peroxidase (Jackson ImmunoResearch); and secondary antibodies for immunofluorescence staining (Invitrogen). RU.521 was purchased from In vivoGen; propidium Iodide, 2’,3’-cGAMP, pan caspase inhibitor (Z-VAD-FMK) and autophagey inhibitor (3-MA and chloroquine) were purchased from Sigma Aldrich; inhibitors of necrosis and ferroptosis (necrostatin-1 and ferrostatin-1) were purchased from Cayman Chemicals. The shRNA clones of ATG5 (TRCN0000330394), cGAS (TRCN0000149984), and STING (TRCN0000134594) were obtained from the RNA Technology Platform and Gene Manipulation Core of the Academia Sinica in Taiwan.
Liu L.C., Shen Y.C., Wang Y.L., Wu W.R., Chang L.C., Chen Y.H., Lee C.C, & Wang S.C. (2022). Growth-promoting function of the cGAS-STING pathway in triple-negative breast cancer cells. Frontiers in Oncology, 12, 851795.
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8

Targeting Cell Death Pathways in PAH

Cited 2 times
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To investigate the contribution of cell death on HMGB1 release and PAH severity, male PAH rats were treated with anti-apoptotic, anti-necroptosis, or anti-necrosis drugs or corresponding vehicles (N=6 in each group). To inhibit apoptosis, rats were injected with a cell-permeable irreversible pan-caspase inhibitor Z-VAD(OMe)-FMK (Biorbyt Ltd, Cambridge, UK, cat# orb181387) in the dose 1 mg/kg i.v. as previously reported28 (link). For necroptosis inhibition, rats were treated with a cell-permeable, potent, and selective blocker of necroptosis Necrostatin-1 (Cayman Chemical, Ann Arbor, MI, cat # 11658), 1 mg/kg i.v.29 (link) Necrosis was attenuated by a cell-permeable inhibitor of necrosis, Necrox-5 methanesulfonate (Cayman Chemical, Ann Arbor, MI, cat# 17278) at the dose 1 mg/kg i.p. as previously published30 (link). Additional two groups of rats were treated with either dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, cat# D2438) in the dose 50 µl of 20% solution per 100g of body weight i.v. as a vehicle for Z-VAD(OMe)-FMK and Necrostatin-1, or with ethanol (Decon Labs., King of Prussia, PA, cat# 2701) 100 µl of 40% solution per 100g of body weight i.p. as a vehicle for Necrox-5. All treatments were initiated on the day of Sugen injection (day 0) and were given daily at the same time of the day during the entire period of study (1 week).
Zemskova M., McClain N., Niihori M., Varghese M.V., James J., Rafikov R, & Rafikova O. (2020). Necrosis-released HMGB1 in the progressive pulmonary arterial hypertension associated with male sex. Hypertension (Dallas, Tex. : 1979), 76(6), 1787-1799.
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9

Pharmacological Screening Library Compounds

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Rifampicin was purchased from AppliChem. Bromopyruvic acid, dexamethasone, gentamicin, staurosporine, and thalidomide were purchased from Sigma-Aldrich. Cyclosporin A, doramapimod, moxifloxacin, necrostatin-1, necrostatin-5, and tacrolimus (FK506) were obtained from Cayman Chemicals. BMS-582949, pamapimod, and Z-VAD-FMK were purchased from Selleckchem. Puromycin dihydrochloride was from Carl Roth (Germany). GR agonist BI653048 was provided by the opnMe platform (Boehringer Ingelheim). The Prestwick chemical library was used for high-throughput screening.
Gräb J., Suárez I., van Gumpel E., Winter S., Schreiber F., Esser A., Hölscher C., Fritsch M., Herb M., Schramm M., Wachsmuth L., Pallasch C., Pasparakis M., Kashkar H, & Rybniker J. (2019). Corticosteroids inhibit Mycobacterium tuberculosis-induced necrotic host cell death by abrogating mitochondrial membrane permeability transition. Nature Communications, 10, 688.
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10

Synthesis and Dissolution of Compounds

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TH34 (3-(N-benzylamino)-4-methylbenzhydroxamic acid) (stock concentration 50 mM) was synthesized by coauthors TH and WS as described previously (Heimburg et al. 2016 (link), 2017 (link)) and was dissolved in DMSO (Sigma-Aldrich). All-trans retinoic acid (ATRA, Sigma-Aldrich, stock concentration 10 mM) was dissolved in ethanol (EtOH, Sigma-Aldrich). Z-VAD-FMK (Biozol, Eching, Germany, stock concentration 100 mM), necrostatin-1 (Cayman Chemical, Tallinn, Estonia, stock concentration 50 mM) and trolox (kindly provided by N. Brady, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA, stock concentration 50 mM) were dissolved in DMSO. N-Acetylcysteine (NAC, Sigma-Aldrich, stock concentration 1 mM) was dissolved in autoclaved Millipore H2O and stored at 4 °C protected from light. If not otherwise specified, compounds were stored at − 20 °C and protected from light.
Kolbinger F.R., Koeneke E., Ridinger J., Heimburg T., Müller M., Bayer T., Sippl W., Jung M., Gunkel N., Miller A.K., Westermann F., Witt O, & Oehme I. (2018). The HDAC6/8/10 inhibitor TH34 induces DNA damage-mediated cell death in human high-grade neuroblastoma cell lines. Archives of Toxicology, 92(8), 2649-2664.
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