Erastin

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.

Erastin and temozolomide were purchased from Cayman Chemical Company (Michigan, USA) and were dissolved in DMSO under sterile conditions to a concentration of 1 and 50 mM, respectively. 1S,3R-RSL3 (RSL3) and Ferrostatin-1 were purchased from Sigma-Aldrich (Taufkirchen, Germany) and were dissolved in DMSO at a concentration of 1 mM.