Erastin

SC line RSC96 was purchased from the cell bank of the China Academy of Sciences (Shanghai, China). The cell line was cultured in DMEM (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% pen/strep (Gibco), and incubated at 5% CO₂ and 37°C.
To establish an HG cell model RSC96 was treated with DMEM containing 100 mM glucose for 48 h as previously described [15 (link)]. Cells were cultured to logarithmic phase and grouped as follows: normal control group (DMEM containing 25 mM glucose), high-glucose group (DMEM containing 100 mM glucose), Erastin (Selleckchem, Houston, USA) group (DMEM containing 25 mM glucose and 2 μM Erastin), and the ferroptosis inhibitor ferrostatin-1 (Fer-1) (Selleckchem) group (DMEM containing 100 mM glucose and 10 μM Fer-1). Erastin, as an inducer of cell ferroptosis, was used as a positive control.

For MTT assays, cells (7500 per well) were seeded in 96-well plates. To determine sensitivity to chemotherapy, after 24 h, cells were treated with different doses of cisplatin (Millipore Sigma) or paclitaxel (ThermoFisher Scientific) for 72 h. To determine the impact of erastin, 24 h after culturing in 96 well plates, cells were treated with cisplatin, erastin (Selleck Chemicals, TX) and ferredoxin(Selleck Chemicals, TX)) either alone or in combination for 72 h. For the RNAi experiment, cells were reverse transfected with negative control siRNA (SIC002) or ABCC1 siRNA (SASI_Hs01_00155530) from SigmaAldrich using Lipofectamine 2000 (ThermoFisher Scientific) as per manufacturer’s protocol. Twenty-four hours post-transfection, cells were treated with erastin and/or ferredoxin for 48 h. For each experiment, the delivery vehicle DMSO was used as the control treatment. The MTT assay was performed after the appropriate treatment as per the manufacturer’s protocol (ATCC) with a few modifications. Briefly, 10 μl of MTT was added to each well and incubated in a 5% CO₂ incubator at 37 °C for 2–4 h. Then 100 μl of detergent was added and the plate was incubated at room temperature in a moist, dark chamber and absorbance was measured at 570 nm using the Biotek Synergy two plate reader Gen5 software (BioTek Instruments).

Cells were seeded onto 96-well plates (2000 cells per cell) and treated with the compounds [RSL3 (Selleck Chemicals), Erastin (Selleck Chemicals), Thrombin (Abcam), Liproxstatin-1 (Selleck Chemicals), Pioglitazone (Sigma-Aldrich), DMSO, Dabigatran (Selleck Chemicals), Trifluoroacetic acid (TFA, Sigma-Aldrich), Darapladib (Selleck Chemicals), NAC (Beyotime)] after plating. Cell viability was assessed at different time points after treatment (24 h unless stated otherwise) using Cell Counting Kit-8 (CCK-8) cytotoxicity assay (Bimake, B34304), as previously described.^{13 (link)} The cell death curve of RSL3 in N27 cells, as well as Thrombin in MDA-MD-231 cells, are shown in Fig. S5.