HT1080 cells expressing ATF3 and empty vector were generated previously [26 (link)]. Retinal pigment epithelial (RPE) cells immortalized with hTERT were obtained from Dr. Todd Waldman [27 (link)]. To generate ATF3-knockout cells, a target sequence (5′-AAAATGATGCTTCAACACCC-3′) spanning the ATF3 start codon was inserted into pSpCas9(BB)-2A-puro, and used to transfect RPE and U2OS cells as described [28 (link)]. ATF3-knockout HCT116 cells were generated using rAAV-mediated homologous recombination as described previously [14 ]. These cells were cultured in media supplemented with 10% fetal bovine serum. We used DMEM for HT1080, RPE, DU145, and U2OS cells, RPMI 1640 for PC3 cells, and McCoy’s 5A for HCT116 cells. Erastin, RSL3, ferrostatin-1, BSO, Z-VAD-FMK, and FIN56 were purchased from Cayman Chemical. N-acetylcysteine (NAC) was purchased from Thermo Fisher Scientific. Other reagents were obtained from Sigma-Aldrich.
Ferrostatin 1
Manufactured by Cayman Chemical
Sourced in United States
Ferrostatin-1 is a chemical compound used in research applications. It functions as a selective inhibitor of ferroptosis, a form of programmed cell death. This product is intended for laboratory use only and its specific applications should be determined by the user.
Automatically generated - may contain errors
Lab products found in correlation
Erastin, by Cayman Chemical (7 mentions)
Necrostatin 1, by Cayman Chemical (5 mentions)
Liproxstatin 1, by Cayman Chemical (5 mentions)
Deferoxamine dfo, by Cayman Chemical (4 mentions)
Ml162, by Cayman Chemical (4 mentions)
Deferoxamine mesylate salt, by Merck Group (4 mentions)
Z vad fmk zvad, by Santa Cruz Biotechnology (3 mentions)
Ml210, by Cayman Chemical (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Vitamin e, by Fujifilm (2 mentions)
Chloroquine, by Merck Group (2 mentions)
Z vad ome fmk, by Cayman Chemical (2 mentions)
Ruxolitinib, by Cayman Chemical (2 mentions)
Mouse ifnγ 485 mi, by R&D Systems (2 mentions)
Gemcitabine, by Selleck Chemicals (2 mentions)
Liperfluo, by Dojindo Laboratories (2 mentions)
Anti ifnγ xmg1.2, by Thermo Fisher Scientific (2 mentions)
L buthionine sulfoximine, by Merck Group (2 mentions)
Siinfekl peptide ova 257 264, by Merck Group (2 mentions)
Jak inhibitor 1, by Cayman Chemical (2 mentions)
24 protocols using ferrostatin 1
1
Generation and Characterization of ATF3 Knockout Cells
2
Evaluating Colorectal and Gastric Cancer Cell Death
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The HT29 human colorectal carcinoma cell line was purchased from Dainihon Pharmacy Co. (Tokyo, Japan). TMK-1 human gastric carcinoma cells were a gift from Professor Wataru Yasui (Molecular Pathology, Hiroshima University, Hiroshima, Japan), and the CT26 murine colon carcinoma cell line was a gift from Professor I. J. Fidler (MD Anderson Cancer Center, Houston, TX, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 °C in 5% CO2.
BBR (25 μM for 48 h unless otherwise specified, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), 5-fluorouracil (5-FU), N-acetyl-L-cysteine (NAC 1 mM), rotenone (0.5 μM) (Sigma-Aldrich Inc., St. Louis, MO, USA), malonate (0.5 mM), vitamin E (20 μM) and vitamin C (1000 μM) (Fujifilm-WAKO Chemicals, Osaka, Japan), Z-VAD-FMK (ZVAD, 20 μM) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ferrostatin-1 (FRS, 2 μM), deferoxamine (DFO, 200 μM) (Cayman Chemicals, Ann Arbor, MI, USA), were purchased from the manufacturers listed.
Cell death was detected using Live-or-Dye™ Fixable Viability staining kit (Cosmobio, Tokyo, Japan). Dead cells were counted by observation of 500 cells. Apoptotic figures were detected by Hoechst 33342 dye (Sigma) apoptotic figures were counted by observation of 500 cells.
BBR (25 μM for 48 h unless otherwise specified, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), 5-fluorouracil (5-FU), N-acetyl-L-cysteine (NAC 1 mM), rotenone (0.5 μM) (Sigma-Aldrich Inc., St. Louis, MO, USA), malonate (0.5 mM), vitamin E (20 μM) and vitamin C (1000 μM) (Fujifilm-WAKO Chemicals, Osaka, Japan), Z-VAD-FMK (ZVAD, 20 μM) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ferrostatin-1 (FRS, 2 μM), deferoxamine (DFO, 200 μM) (Cayman Chemicals, Ann Arbor, MI, USA), were purchased from the manufacturers listed.
Cell death was detected using Live-or-Dye™ Fixable Viability staining kit (Cosmobio, Tokyo, Japan). Dead cells were counted by observation of 500 cells. Apoptotic figures were detected by Hoechst 33342 dye (Sigma) apoptotic figures were counted by observation of 500 cells.
3
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%.
4
Hepatocyte Viability Assay with APAP
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Hepa1 and Huh7 hepatocytes were grown in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum (FBS), 100 units mL−1 penicillin, and 100 µg mL−1 streptomycin. Primary hepatocytes were isolated from mice using type II collagenase (Worthington Biochem, Lakewood, NJ), as described.(28 (link)
) Primary hepatocytes were grown on William’s medium E (Sigma) supplemented with 2% FBS, 100 units mL−1 penicillin, and 100 µg mL−1 streptomycin and transduced with β‐galactosidase (β‐gal) or NIK adenoviral vectors. After 12‐14 hours of growth, hepatocytes were treated with APAP for 2‐24 hours in the presence or absence of NAC, ferrostatin‐1 (Item No. 17729, CAS No. 347174‐05‐4; Cayman Chemicals), or liproxstatin‐1 (Item No. 17730 CAS No. 950455‐15‐9, Cayman Chemicals). Hepatocyte viability was measured using colorimetric 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assays (DOT Scientific Inc., Burton, MI).
) Primary hepatocytes were grown on William’s medium E (Sigma) supplemented with 2% FBS, 100 units mL−1 penicillin, and 100 µg mL−1 streptomycin and transduced with β‐galactosidase (β‐gal) or NIK adenoviral vectors. After 12‐14 hours of growth, hepatocytes were treated with APAP for 2‐24 hours in the presence or absence of NAC, ferrostatin‐1 (Item No. 17729, CAS No. 347174‐05‐4; Cayman Chemicals), or liproxstatin‐1 (Item No. 17730 CAS No. 950455‐15‐9, Cayman Chemicals). Hepatocyte viability was measured using colorimetric 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assays (DOT Scientific Inc., Burton, MI).
5
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).
6
Ferroptosis Pathway Modulation Assay
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TVB-3664 (FASNi) or the inactive isomer (TVB-2632) were from Sagimet Biosciences (San Mateo, CA). ML162 (#20455) or Ferrostatin-1 (#17729) were from Cayman. ARS-1620 (HY-U00418) and Liproxstatin-1 (HY-12726) were from MedChemExpress. 16:0–18:1 PC (hereafter PC #850457P), Sodium palmitate (#P9767) and N-Acetyl-L-cysteine (#A7250) were from Millipore-Sigma. CellROX™ Green Reagent (#C10444) was from Thermo Fischer Scientific.
7
Chemical Compounds for Cell Death Study
8
Cell Death Inhibitor Assay in H9c2 Cells
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For the cell death inhibitor assay, H9c2 cells were treated with human recombinant HMGB1 (40 μg/mL, Biolegend, San Diego, CA, USA) for 48 h, with or without the addition of cell death inhibitors. The following inhibitors were utilized: N-acetyl-L-cysteine (NAC, 1 mM, Sigma, St. Louis, MO, USA), Z-VAD-FMK (ZVAD, 20 μM, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ferrostatin-1 (FRS, 2 μM), deferoxamine (DFO, 200 μM, Cayman Chemicals, Ann Arbor, MI, USA), and 4-phenylbutyric acid (4PBA, 200 μM, WAKO). Additionally, chloroquine (100 µM, WAKO) was employed to inhibit autophagy.
9
Ferroptosis-associated Compounds Evaluation
10
Compounds Screening for Cell Viability
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Cells were seeded in 96-well plates (Corning 3917, 3125–6250 cells per well) and treated with compounds 24 h after plating. Compounds were purchased from Cayman Chemical with the exception of stearic acid, oleic acid, adrenic acid, linolenic acid, linoleic acid, docosahexaenoic acid, conjugated linoleic acid (16413), bovinic acid, arachidonic acid, Z-VAD-FML, and (+)-α-tocopherol (T3634), which were purchased from Sigma Aldrich. RSL3 was purchased from Selleckchem, catalpic acid, α-calendic acid, β-calendic acid, and β-ESA were obtained from Larodan Fine Chemicals, and punicic acid was purchased from Matreya LLC. Initial aliquots of ferrostatin-1, deferoxamine, and ML162 were generous gifts of the Dixon Lab (Stanford) and additional quantities were purchased from Cayman Chemical. The inhibitor of FSP19 (link) was purchased from ChemBridge Corp. Staurosporine was purchased from LC Sciences. Cell viability was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer’s instructions. Luminescence was measured on an EnSpire Alpha (Perkin Elmer) using the integrated software package. Data were normalized to vehicle-treated or sensitizing agent-alone controls and IC50 curves were produced with GraphPad Prism (Version 8).
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