Erastin

MDA-MB-231 and HCC1937 cells were seed in 96-well plates for each cell line (TNBC and non-TNBC, ~13 cell lines) at 4000 cells per well, and cell viability assay was conducted with CCK-8 kit after combinational treatment of CB1 antagonists with ferroptosis inducers (erastin, RSL3, ML210, FIN56, CIL56) for 72 h according to the manufacturer’s instructions (Dojindo Laboratories, Japan). For cell death rescue experiments, the effects of various cell death pathway inhibitors were calibrated for each cell line for doses and effects. Ferroptosis inhibitors (Ferrostatin-1, GSH, and Deferoxamine), and cell death inhibitors were used to inhibit apoptosis (zVAD-FMK), necrosis (Necrostatin-1), autophagy (3-Methyladenine) at indicated concentrations 1 h before the treatment with combination therapy.

The impacts of H₂O₂, Erastin, and RSL3 on the cell viability of ESCs were measured by the Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan) method. Cells were inoculated in 96-well plates, and 500 µM H2O2 (Sigma), 20 µM Erastin (MedChemExpress, Shanghai, China), and 5 µM RSL3 (MedChemExpress) were added into plates. After 6 h, the culture solution was discarded. Cells were then incubated in 100 μl fresh culture solution and 10 μl CCK-8 at 37°C for 2 h. Next, the absorbance of the solution at 450 nm was measured by Spark (Tecan, Salzburg, Austria) to calculate the cell viability of ESCs.