P06 07300

Cell lines ES2 (CRL-1978), OVCAR-3 (HTB-161) and OVCAR-8 (CVCL-1629) were obtained from American Type Culture Collection (ATCC). Cell line A2780 sensitive (93112519) and A2780 cisplatin resistant (93112517) were obtained from Sigma Aldrich. Cells were maintained at 37 °C in a humidified 5% CO₂ atmosphere. ES2, OVCAR3 and OVCAR8 cell lines were cultured in DMEM (41965–039, Gibco, Life Technologies) supplemented with 1% FBS (S 0615, Merck), 1% antibiotic-antimycotic (AA) (P06–07300, PAN Biotech). A2780 parental and A2780 cisR cells were cultured in RPMI 1640 (BE12-167F, Lonza) supplemented with 0.58 g/L of L-glutamine, 1% FBS (S 0615, Merck) and 1% antibiotic-antimycotic (AA) (P06-07300, PAN Biotech).
Cells were exposed to 0.4 mM L-Cysteine (102839, Merck) and/or exposed to hypoxia-induced conditions with 0.1 mM cobalt chloride (C8661, Sigma-Aldrich). Cobalt is a hypoxia mimicking agent used in in vivo^{53 (link)} and in vitro^{54 –56 (link)} studies. Chemically, CoCl₂ reacts with oxygen impairing its dissolution and oxygenation of aqueous solutions⁵⁷ and is a way of impairing the availability of oxygen in culture media.
Prior to any experiment, cells were synchronized under starvation (culture medium without FBS) for 8 h at 37 °C and 5% CO₂.

Three different batches corresponding to three different-original donors of Human umbilical vein endothelial cells (HUVECs: CRL-1730, ATCC) were used. HUVECs were cultured in Endothelial Cell Growth Basal Medium-2 (EBM-2: CC-3156, Lonza, Bioscience) supplemented with EGM-2 SingleQuots Supplements (CC-4176, Lonza, Bioscience). All experiments were performed until the passage 10. Triple-negative breast cancer (MDA-MB-231: HTB-26™, ATCC) were used as tumor models, being cultured in Dulbecco’s Modified Eagles Medium (DMEM) (41965-039, Gibco, Life Technologies), supplemented with 10% fetal bovine serum (FBS) (S 0615, Merck), 1% Antibiotic-Antimycotic (AA; P06-07300, PAN Biotech) and 50 µg/mL Gentamicin (15750-060, Gibco, Life Technologies). Cell cultures were maintained at 37°C in a humidified environment of 5% CO₂. Cells were detached with 0.05% Trypsin-EDTA 1 × (25300-054, Invitrogen, Thermo Fisher Scientific) at 37°C for approximately 5 min and split to new plates according to the experimental procedures.
Regarding experimental conditions, cells were cultured with 15 μM hydrogen peroxide (H₂O₂; 1.07210.0250, Merck), as a ROS generator, 1.5 μM Erastin (E7781, Sigma) as a ferroptosis inducer, 100 μM Propranolol (P8688, Sigma Aldrich) and 160 and 200 μM SeChry@PURE_G4, for 6 and 16 h.

Cell lines from OCCC (ES2; CRL-1978) and HG-OSC (OVCAR-3; HTB-161) were obtained from American Type Culture Collection (ATCC). Cells were maintained at 37°C in a humidified 5% CO₂ atmosphere, and cultured in Dulbecco’s Modified Eagle medium (DMEM, 41965-039, Gibco, Life Technologies), containing 4.5 g/L of D-glucose and 0.58 g/L of L-glutamine, 1% FBS (S 0615, Merck), 1% antibiotic-antimycotic (P06-07300, PAN Biotech) and 0.1% gentamicin (15750-060, Gibco, Life Technologies). Cells were exposed either to 0.402 mM L-cysteine (102839, Merck) and/or to hypoxia induced with 0.1 mM cobalt chloride (CoCl₂) (C8661, Sigma-Aldrich) as previously (Nunes et al., 2018a (link),b (link)).
Prior to any experiment, cells were synchronized under starvation (culture medium without FBS) for 8 h at 37°C and 5% CO₂.