Ferrostatin

MCF7 (from ATCC) and previously established neuT and neuTemt cells [32 (link)] were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin. MCF10DCIS.com cells (obtained from Asterand) and DCIS-Snai1-ER cells [33 (link)] were maintained in DMEM/F12 supplemented with 5% horse serum. Glucose-, pyruvate- and glutamine-free DMEM was obtained from Gibco (catalog A14430). Glucose and glutamine were added to reconstitute low-glucose media. Metformin, phenformin, 2DG, BAY-876, disulfiram, ferrostatin, and necrostatin were purchased form Cayman Chemical.

Cells were seeded at 2000 cells per well on a 96-well plate and were grown overnight at 37 °C. The following day, cells were treated with the following drugs at indicated concentrations for 72 h for IC₅₀ assays: erastin (Cayman Chemicals), RSL3 (Apex Bio), TBH(Sigma), nutlin-3a (Calbiochem), cisplatin (Acros Organics), Etoposide (Sigma), Camptothecin (Cayman Chemicals), and doxorubicin (Cell Signaling). Because fetal bovine serum and culture media can contain significant levels of glutathione, pyruvate, and other agents that can influence sensitivity to ferroptosis (10 (link)), all ferroptosis sensitivity assays were performed in low serum/low glucose (2% fetal bovine serum, DMEM low glucose (Corning Cellgro, 1 g/l glucose)). For rescue assays, cells were pretreated with ferrostatin (Cayman Chemicals) or liproxstatin (Sigma) for 30 min prior to treatment with RSL3 for 24 h. For lipid droplet assays, cells were pretreated overnight with oleic acid (Sigma, O3008), the DGAT2 inhibitor PF-06424439 (Sigma, PZ0233), or vehicle (fatty-acid–free BSA), and viability was assayed using Trypan Blue (Corning, 25-900-CI) after 72 h. At assay endpoint, cells were treated with Alamar blue (Life Technologies Dal1025) for 3 h at 37 °C and absorbance was read using a SynergyHT plate reader (BioTek). GraphPad Prism software was used to perform the data analysis.