Erastin

Manufactured by Merck Group

Sourced in United States, Germany, Morocco, China

Erastin is a chemical compound used as a laboratory research tool. It functions as a ferroptosis inducer, capable of triggering a specific form of regulated cell death. The core function of Erastin is to disrupt cellular processes related to iron metabolism and lipid peroxidation. Detailed information about intended use or applications is not provided.

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Erastin (E7781) (3 citations)

128 protocols using erastin

Intervertebral Disc Degeneration in Mice

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Fifty C57BL/6 male mice (aged 8 weeks) provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., (Beijing, China) were raised under specific pathogen-free conditions (22°C, 50%-55% humidity, and 12 h light/dark cycle). The specific procedures were performed as previously reported method [18 (link)]. The Co6-Co7 discs were collected at week 6 and 12 after surgery from the mice for subsequent experiments.
Mice were divided into control group (n = 10) and IDD group (n = 40). Next, IDD mice (n = 40) were injected with PBS, BMSC-EVs (200 μg), ferroptosis inducer Erastin (50 mg/kg, EMD Millipore Corp., Billerica, MA), or Erastin+EVs (200 μg BMSC-EVs and 50 mg/kg Erastin) via tail vein (n = 10). The mice were injected every 2 days until they were euthanized.

Yu X., Xu H., Liu Q., Wang Y., Wang S., Lu R., Jiang Y., Kang H, & Hu W. (2022). circ_0072464 Shuttled by Bone Mesenchymal Stem Cell-Secreted Extracellular Vesicles Inhibits Nucleus Pulposus Cell Ferroptosis to Relieve Intervertebral Disc Degeneration. Oxidative Medicine and Cellular Longevity, 2022, 2948090.

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Investigating Erastin-Induced Cell Death in SFXN2-KO Cells

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Control and SFXN2-KO cells were seeded in a 96-well plate at a density of 5000 cells/well. The following day, cells were treated with 10 μM erastin (Sigma) and incubated for 24 h. Cell viability was monitored using WST-8 reagent (Dojindo) according to the manufacturer’s instructions. Absorbance was measured at 405 nm. To investigate cell death, control and SFXN2-KO cells were seeded in a 96-well plate at a density of 10,000 cells/well. The following day, cells were treated with 10 μM erastin (Sigma) and incubated for 24 h. Cell death was assessed by the LDH assay and trypan blue staining in the same well. The culture medium in each well was transferred to a fresh 96-well plate. The amount of LDH released into the culture medium was measured using a Cytotoxicity LDH Assay Kit-WST (Dojindo) according to the manufacturer’s instructions. Cells were stained with trypan blue (Sigma) and examined under a microscope (Olympus) equipped with a CCD camera.

Mon E.E., Wei F.Y., Ahmad R.N., Yamamoto T., Moroishi T, & Tomizawa K. (2018). Regulation of mitochondrial iron homeostasis by sideroflexin 2. The Journal of Physiological Sciences, 69(2), 359-373.

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Erastin-Loaded Lipid Nanoparticle Synthesis

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Precisely weighed 120 mg of l-α-phosphatidylcholine (Solarbio, Beijing, China), 40 mg of cholesterol (Solarbio), 20 mg of DSPE-PEG-FA (Shanghai Ponsure Biotechnology, China, Shanghai), and 3 mg of erastin (Sigma-Aldrich, MO, USA) were fully dissolved in 5 mL chloroform and mixed with 3 mL of 1 mg/mL erastin chloroform solution. Chloroform was then removed using a rotary evaporator to form a dry-lipid film at 35 °C under uniform speed. MT1DP (300 nM) dissolved in phosphate-buffered saline (PBS) (pH 7.4, 5 mL) was added to the lipid film and hydrated for 60 min. After the hydration was completed, the cells were sonicated with an ultrasonic cell disruptor and passed through a polyethersulfone membrane with a membrane pore diameter of 450 and 220 nm to decrease the particle size.

Gai C., Liu C., Wu X., Yu M., Zheng J., Zhang W., Lv S, & Li W. (2020). MT1DP loaded by folate-modified liposomes sensitizes erastin-induced ferroptosis via regulating miR-365a-3p/NRF2 axis in non-small cell lung cancer cells. Cell Death & Disease, 11(9), 751.

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Ferroptosis Inhibition in Osteoblasts

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Cell culture and treatment
The hFOB1. Erastin (Sigma-Aldrich) was used as positive control for cell ferroptosis. Deferoxamine (DFO, Sigma-Aldrich) and Ferrostatin-1 (Ferr-1, Sigma-Aldrich) were used to inhibit Fe or Erastin induced cell death. All samples were collected for relative assays.
Cell proliferation assay hFOB1.19 osteoblast cells were seeded (10 3 cells per well) in 96-well plates. After cells were treated with Erastin or Fe or/and Ferr-1 or/and DFO, a solution containing fresh medium (90 μl) and CCK-8 reactant (10 μl) (Dojindo) was added to each well, and cells were incubated at 37 °C for 1.5 h in the dark followed by addition of stop buffer. Absorbance at 450 nm was then measured using an enzyme-labelled instrument (Thermo). All operations were performed in accordance with the manufacturer's instructions.

Zhang H., Wang A., Li G., Zhai Q., Huang Z., Wang X., Cao Z., Liu L., Liu G., Chen B., Zhu K., Xu Y, & Xu Y. (2023). Osteoporotic bone loss from excess iron accumulation is driven by NOX4-triggered ferroptosis in osteoblasts. Free radical biology & medicine, 198.

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Erastin-induced cell death in HT22 cells

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HT22 cells (kindly provided by David Schubert, Cellular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA) were grown in Dulbecco’s modified Eagle medium (DMEM, Capricorn Scientific GmbH, Ebsdorfergrund, Germany) supplemented with 10% heat-inactivated fetal calf serum (Merck KGaA, Darmstadt, Germany), 100 U/mL penicillin, 100 mg/mL streptomycin (Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and 2 mM L-glutamine (Merck KGaA, Darmstadt, Germany). To induce cell death, erastin (Calbiochem^®, Merck KGaA, Darmstadt, Germany) was added to the medium for the indicated amount of time (8–16 h).

Rabenau M., Dillberger B., Günther M., Krippner S., Butterweck V., Boonen G., Drewe J., Eckert G.P, & Culmsee C. (2021). Cimicifuga racemosa Extract Ze 450 Re-Balances Energy Metabolism and Promotes Longevity. Antioxidants, 10(9), 1432.

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Ferroptosis Induction and Inhibition Compounds

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Menaquinone-4 (V9378), phylloquinone (V3501), menadione (M5625), MK4 epoxide (75618), vitamin K3 epoxide (51455), α-TOC (T3251), warfarin (A2250), warfarin sodium (PHR1435), dicoumarol (M1390), l-buthionine sulfoximine (BSO; B2515) N-acetyl-l-cysteine (A7250), lipopolysaccharide (LPS; L2880) and MCC950 (5381200001) were purchased from Sigma-Aldrich. Ferrostatin-1 (Fer1; 17729), RSL3 (19288), FINO₂ (25096), ML162 (20455), ML160 (23282) and staurosporine (81590) were purchased from Cayman. The following chemicals were obtained as indicated: erastin (329600, Merck Millipore), 17-AAG (A10010, Adqoo), l-glutamate (16911-22, Nacalai tesque), menadiol (M323135, TRC), iFSP1 (8009-2626, ChemDiv), liproxstatin-1 (Lip1, S7699, Selleckchem), BV-6 (S7597, Selleckchem), Trolox (56510, Fluka), recombinant mouse TNF (PMC3014, Thermo Fishier), nigericin (N1495, Thermo Fisher), zVAD-FMK (ALX-260-02, Enzo Life Sciences) and Nec 1s (2263, BioVision).

Mishima E., Ito J., Wu Z., Nakamura T., Wahida A., Doll S., Tonnus W., Nepachalovich P., Eggenhofer E., Aldrovandi M., Henkelmann B., Yamada K.I., Wanninger J., Zilka O., Sato E., Feederle R., Hass D., Maida A., Mourão A.S., Linkermann A., Geissler E.K., Nakagawa K., Abe T., Fedorova M., Proneth B., Pratt D.A, & Conrad M. (2022). A non-canonical vitamin K cycle is a potent ferroptosis suppressor. Nature, 608(7924), 778-783.

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Evaluating Cell Proliferation and Viability

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Cell proliferation was evaluated by using electrical impedance monitoring, through the xCELLigence Real-Time Cell Analysis (RTCA; Roche Diagnostics) system.
When cells proliferate, the current flow between the microelectrodes, placed in the bottom surface of each well of a 96-well plate, is impeded. The impedance of this electron flow is reported as arbitrary cell index-values. For this assay, CTR and KO MEF cells were seeded in a 96-well plate (6000 cells/well) and treated or not with erastin (0.7 µM; Calbiochem).
Metabolic activity as an indicator for cell viability was assessed through the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. In viable and metabolically active cells, MTT is reduced to a purple formazan. CTR and KO MEF cells have been seeded in 96-well plates (2000 cells/well) and treated with erastin (0.7 µM) for 8 h. Subsequently, MTT (0.5 mg/mL; Merck) was added for 1 h at 37 °C for the purple formazan production. Absorbance was measured at 570–630 nm with FLUOstar (BMG Labtech).

Kepser L.J., Khudayberdiev S., Hinojosa L.S., Macchi C., Ruscica M., Marcello E., Culmsee C., Grosse R, & Rust M.B. (2021). Cyclase-associated protein 2 (CAP2) controls MRTF-A localization and SRF activity in mouse embryonic fibroblasts. Scientific Reports, 11, 4789.

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Ferroptosis Regulators Protocol

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Lip-1 (Selleckchem, cat. no. S7699), doxycycline (Dox; Sigma, cat. no. D9891), RSL3 (Cayman, cat. no. 19288), BSO (Sigma, cat. no. B2515), iFSP1 (ChemDiv, cat. no. 8009-2626), icFSP1 (ChemDiv, cat. no. L892-0224 or custom synthesis by Intonation Research Laboratories), erastin (Merck, cat. no. 329600), ML210 (Cayman, cat. no. Cay23282-1), FIN56 (Cayman, cat. no. Cay25180-5), FINO2 (Cayman, cat. no. Cay25096-1), deferoxamine mesylate salt (DFO; Sigma, cat. no. 138-14-7), ferrostatin-1 (Fer-1; Sigma, cat. no. SML0583), zVAD-FMK (zVAD; Enzo Life Sciences, cat. no. ALX-260-02), necrostatin-1s (Nec-1s; Enzo Life Sciences, cat. no. BV-2263-5), MCC950 (Sigma, cat. no. 5381200001), olaparib (Selleckchem, cat. no. S1060), staurosporine (STS; Cayman, cat. no. 81590), recombinant human tumour necrosis factor (TNF; R&D Systems, cat. no. NBP2-35076), Smac mimic (BV-6; Selleckchem, cat. no. S7597), nigericin (Thermo Fisher, cat. no. N1495), IMP-1088 (Cayman, cat. no. Cay25366-1) and lipopolysaccharide (LPS; Sigma, cat. no. L2880) were used in this study.

Nakamura T., Hipp C., Santos Dias Mourão A., Borggräfe J., Aldrovandi M., Henkelmann B., Wanninger J., Mishima E., Lytton E., Emler D., Proneth B., Sattler M, & Conrad M. (2023). Phase separation of FSP1 promotes ferroptosis. Nature, 619(7969), 371-377.

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Heme Oxygenase-1 Regulation Analysis

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Erastin (EMD Millipore Corporation, USA), Znpp-IX (ENZO Life Science, Plymouth Meeting, PA), Carbon monoxide-releasing molecule (CORM) (Sigma-Aldrich, St Louis, MO), hemin, bilirubin (Frontier Scientific, Inc, USA), and biliverdin (MP Biomedicals, LLC, France) were used. Deferoxamine (DFO) and cycloheximide were purchased from Sigma-Aldrich (St. Louis, MO). Anti- HO-1 was purchased from StressGen Biotechnologies Inc. (Victoria, BC, Canada). Anti-b-actin was purchased from Sigma-Aldrich (St Louis, MO). Horseradish peroxidase (HRP) conjugated goat anti-rabbit or goat anti-mouse IgG was from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Kwon M.Y., Park E., Lee S.J, & Chung S.W. (2015). Heme oxygenase-1 accelerates erastin-induced ferroptotic cell death. Oncotarget, 6(27), 24393-24403.

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Screening Chemical Inhibitors for Cell Death

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U0126 was obtained from Cayman Chemical (Ann Arbor, MI). Fer-1, MEK inhibitor I, (Z)-4-hydroxytamoxifen (Tamoxifen), DFO, Trolox, and 3-methyladenine (3-MA) and erastin were obtained from Merck Millipore (Burlington, MA). RSL3, Z-VAD-FMK, and Ulixertinib were obtained from MedChemExpress (Monmouth Junction, NJ). Necrostatin 2 racemate (Nec-1s), pluripotin, and AZD0364 were obtained from Selleck Chemicals (Huston, TX). The chemical inhibitor library for several enzymes (SCADS inhibitor kits) was kindly gifted by the Molecular Profiling Committee.

Tsuruta K., Matsuoka M., Harada S., Enomoto A., Kumagai T., Yasuda S., Koumura T., Yamada K.I, & Imai H. (2023). Slowly progressive cell death induced by GPx4-deficiency occurs via MEK1/ERK2 activation as a downstream signal after iron-independent lipid peroxidation. Journal of Clinical Biochemistry and Nutrition, 74(2), 97-107.

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