Ferrostatin

Nuclight Red cell lines were seeded at 20,000 cells in a 96-well plate (SYO-WT, SYO-OE, Fuji WT, Fuji OE, HTB-93-WT, and HTB-93-KDs) 1 day before assay. Cells were treated with either 25 μmol/L G6PDi-1 (72 hours), 2 mmol/L BSO (24 hours), 1.2 μmol/L D9 (12 hours), 250 μmol/L deferoxamine (5 hours), erastin (dosage indicated per cell line 48 hours), or 1 μmol/L ACXT-3102 (5 hours). For rescue of erastin treatment with lipophilic antioxidants or ferrostatin, the dosages were as follows: 200 μmol/L Trolox, 100 μmol/L a-tocopherol, and 10 μmol/L ferrostatin. Erastin rescue experiments were treated for 72 hours. Phenol red-free media containing drug concentration as well as 50 nmol/L of YOYO-1 iodide (Y3601, Thermo Fisher Scientific Inc) cell death marker replaced growth media. Cell death was measured via the YOYO-1 iodide probe, and live-cell analysis was measured using Nuclight Red (4717, Sartorius). Cell death was analyzed using IncuCyte FLR imaging system software (Sartorius). For quantification of death, YOYO-1 florescence was normalized to total counts for each respective well. The drugs utilized in these experiments were obtained as follows: BSO (14484, Cayman Chemical), D9 (5921, Tocris), deferoxamine (S5742, Selleckchem), Trolox (93510, Millipore-Sigma), a-tocopherol (1667600, Millipore-Sigma), and ferrostatin (S7243, Selleckchem).

Ectopic expression and gene silencing experiments were conducted to determine the effects of circRHOT1 and the molecular mechanism. The overexpression vectors of circRHOT1, KAT5, and GPX4, and shRNAs (i.e., shcircRNOT1, shKAT5, and shNC) were synthesized by RiboBio (China). Cell transfection was performed using Lipofectamine 2000 (Invitrogen, USA) in accordance with the manufacturer’s instructions. Next, to induce ferroptosis, the cells were treated with a ferroptosis inducer erastin or inhibitor ferrostatin (5 µM; Selleck, USA) for 24 hours.

DMEM High Glucose (Gibco), RPMI 1640 (Gibco), Trypsin 0.05% (Gibco), penicillin/streptomycin 1X (Gibco), Glutamine (Gibco), Matrigel (Corning), Polybrene (Santa Cruz Biotechnology), Polyethylenimine (Sigma-Aldrich), BRD2 antibody (Bethyl Laboratories), β-actin (Cell Signaling Technology), Osimertinib (MedchemExpress, Selleck), CID compound (Takara 635088), Doxycycline (Sigma), Alisertib (Selleck), Trametinib (Medko), Dabrafenib (MedchemExpress), Crizotinib (Selleck), AMG510 (Selleck), RSL3(Selleck), NEO2734(Selleck), ARV-771(Selleck), CC90010 (Selleck), ABT-263 (Selleck), Ferrostatin (Selleck), Z-VAD-FMK (Selleck), N-acetylcysteine amide (Sigma).