Ferrostatin 1

Human osteosarcoma cell lines – U2OS, MG63 and HOS – were purchased from ‘Biological Resource Center ICLC Cell bank, Core facility IRCCS Ospedale Policlinico San Martino, Genova (IT)’, were maintained in Dulbecco’s modified Eagle’s medium (DMEM, EuroClone), supplemented with 10% fetal bovine serum (FBS, EuroClone), 2 mM L-glutamine (Merck), 1% penicillin/streptomycin (Merck), at 37 °C in a humidified incubator with 5% CO₂. Mycoplasma testing was routinely performed each month by using the Venor®GeM Classic (Minerva-BiolAbs;Berlin, GE)
Cells were treated with 0.5 µM RSL3 (Merck), 10 µM Ferrostatin-1 (Merck), 6 µM iFSP1 (BioVision), 50 nM Brustatol (Cayman Chemicals); 10 µM MPA (Merck), or 10 µM Pifithrin (Merck), as indicated.

Erastin (S7242), Z-VAD-FMK (S7023), 3-methyladenine (S2767) and disulfiram (S1680) were obtained from Selleck. Ferrostatin-1 (SML0583-5MG) was obtained from Merck.

SFN, necrostatin-1, ferrostatin-1, and N-acetylcysteine were purchased from Merck Millipore (Darmstadt, Germany); N-Benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl ketone (Z-VAD-FMK) was obtained from Calbiochem, EMD Millipore Corp. (Billerica, MA, USA); necrosulfonamide was purchased from Tocris Bioscience (Bristol, UK); (1S,3R)-RSL3 (RSL3) and SM164 were obtained from Cayman Chemical (Ann Arbor, MI, USA); tumor necrosis factor α (TNF-α) was purchased from PeproTech GmbH (Hamburg, Germany).
Exponentially growing cells were used for all experiments. Cells were treated with increasing concentrations of SFN (0–50 μM) for 1, 3, 6, 9, 16, or 24 h, depending on the experimental conditions. To evaluate the induction of non-canonical cell death pathways, cells were pre-treated for 1 h with different chemical inhibitors and then treated with SFN for 24 h. The following inhibitors were used: the pan-caspase inhibitor Z-VAD-FMK (50 μM), the RIP1 inhibitor necrostatin-1 (50 μM), the MLKL inhibitor necrosulfonamide (1 μM), and the inhibitor of ROS generation and lipid peroxidation ferrostatin-1 (1 μM), to inhibit apoptosis, necroptosis, and ferroptosis, respectively. TNF-α (50 ng/mL) + SM164 (500 nM) + Z-VAD-FMK (50 μM) were used as positive control for necroptosis induction. RSL3 (0.2 μM) was used as a positive control for ferroptosis induction.