Erastin e7781

Suffruticosol C and resveratrol were isolated from the seeds of the tree peony. The concentration was chosen based on previous studies [27 (link),28 (link),31 (link),32 (link)] and considering that suffruticosol C is a trimer of resveratrol. The secondary antibodies and Erastin (E7781) were provided by Sigma-Aldrich (St. Louis, MO, USA). RSL3 (1219810-16-8) was obtained from Cayman (Ann Arbor, MI, USA), and Bafilomycin A1 (S1413) was obtained from Selleck Chemicals (Houston, TX, USA). PBS and trypsin were obtained from HyClone (Logan, UT, USA). The RPMI 1640 and DMEM media, streptomycin, β-mercaptoethanol, penicillin, and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). The TB Green qRT-PCR kit (RR820A), PrimeScript™ RT reagent Kit (RR047A) and TRIzol were provided by Takara (Dalian, China). The primary antibodies used are listed as follows: Bcl-2 (12789-1-AP), Bax (50599-2-Ig), LC3II (18725-1-AP), p62 (18420-1-AP), and Actin (20536-1-AP) were purchased from Proteintech (Rosemont, IL, USA); S6 (2217S), p-S6 (4858S), AKT (9272), pT389-S6K (9234S/L), S6K (9202S), and Cleaved Caspase-3 (D175) were purchased from Cell Signaling Technology (Danvers, MA, USA).

LDH is a marker found in the cytoplasm. When the cell membrane is broken, this molecule is released from the cytoplasm of the cell into the culture medium. The amount of LDH leaked was determined using an LDH cytotoxicity detection kit (CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega)). Approximately 6000 HEI-OC1 and Nrf2 KO cells/well were cultivated separately in 96-well plates overnight and then treated with or without cisplatin. According to the manufacturer's instructions, 100 μL of fresh reaction solution was added to each well for 30 min of incubation at room temperature (RT) after the different treatments, and a microplate reader (BioTek, Epoch) was used to estimate LDH release by measuring the absorbance at 492 nm. The classical ferroptosis inducers 1S,3R-RSL3 (#SML2234) and erastin (#E7781) were purchased from Sigma.

Oleuropein was extracted from olive leaves of the Coratina cultivar of Olea europaea L. as reported by Procopio et al. [50 (link)]. Briefly, olive leaves were dried, milled, and extracted in an Anton Paar Synthos 3000 MW Oven, at 800 W (P-controlled mode) for 10 min, using water as solvent. Then, the leaves were filtered, the solution was dried under pressure, and acetone was added to the mixture. The solid residue was eliminated by filtration, the solution was evaporated under reduced pressure and the crude was purified by flash chromatography on silica cartridges (CH₂Cl₂/MeOH 8:2). Oleuropein was obtained at an HPLC purity of 98%. Analytical data of the pure oleuropein were compared with data reported in the literature. Erastin (E7781) was purchased from Sigma-Aldrich, St. Louis, MO, USA. For oleuropein and erastin treatments, HEY and MCF7 cells were plated at a density of 5.0 × 10⁵ cells/well in a 6-well plate in a complete medium. The next day, cells were treated with oleuropein (at 100, 200, and 400 µM for 24 h) and erastin at 2.5 µM for 24 h. The used OLE concentration and times of exposure agreed with previous literature studies [20 (link),22 (link),40 (link),47 (link)].