Necrosulfonamide

Erastin (a ferroptosis inducer) and Ferrostatin-1 (a ferroptosis inhibitor) were obtained from Selleck Chemicals (Houston, TX, USA). ZVAD-FMK (an apoptosis inhibitor) and necrosulfonamide (a necroptosis inhibitor) were purchased from Sigma (St. Louis, MO, USA).

HCT-116 were from ATCC. HCT116 (Bax/Bak)^−/− cells were a gift from R. Youle (NIH, Bethesda, MD), MEF and MEF (Bax/Bak)^−/− cells were a gift from S. Oakes (University of California, San Francisco, CA). Cells were authenticated and regularly mycoplam tested. TRAIL was provided by C.R. Rodrigues dos Reis (University of Groningen, Netherlands). Tunicamycin was purchased from Alexis (San Diego, CA), bortezomib from Millenium Pharmaceuticals (Cambridge, MA), MG-132 from Biomol (Plymouth Meeting, PA, USA), MnTBAP, 3-MA, 5-FU, Suc-LLVY, cisplatin, and necrostatin-1 from Calbiochem, z-VAD-fmk from Bachem (St. Helens, UK), Hoechst 33342 from Immunochemistry (UK), Aggresome Staining Kit, and TRAIL-R1:Fc (ALX-522-004-C050), TRAIL-R2:Fc (ALX-522-005-C050), and TNF-R1:Fc (ALX-522-013-C050) neutralizing antibodies from Enzo Life Sciences (Exeter, UK), Coomassie PLUS (Bradford) protein assay kit and Pierce BCA protein assay kit from Thermo Fisher Scientific, RPMI1640 medium, Q-VD-OPh, rotenone, epoxomicin, propidium iodide, ferrostatin-1, necrosulfonamide, FBS and all other chemicals were purchased from Sigma-Aldrich. Q-VD-OPh was added in concentrations of up to 20–50 µM to fully prevent caspase processing and substrate cleavage, where required [6 (link), 7 (link)].

Human recombinant TNFα (R&D Systems, Cat#210-TA-005); Recombinant mouse mTNFα (Cell sciences, Cat# CRT192C); Cycloheximide (Sigma, Cat# C-6255); SM-164 (Selleckchem, custom-synthesized); Birinapant (Selleck Chemicals, Cat# S7015); zVAD (Selleck Chemicals, Cat# S8102); Nec-1s (7-Cl-O-Nec-1) (Selleck Chemicals, Cat# S8641); GSK-872 (Sigma, Cat# 5303890001); Necrosulfonamide (Sigma, 480073); LPS (Sigma, Cat# L4391); IDN-6556 (Selleck Chemicals, Cat# S7775); Poly(I:C) (InvivoGen, Cat# tlrl-pic-5); 5Z-7-Oxozeaenol (Sigma, Cat# O9890); 5-fluorouracil (Selleck Chemicals, Cat# S1209); Necrosulfonamide (Selleck Chemicals, Cat# S825); and ELISA Kit for Human TNFα (BioLegend, Cat# 430206).

Human ovarian cancer SKOV3 and OVCA429 cells were maintained in RPMI 1640 (SKOV3 cells) or DMEM (OVCA429 cells) medium supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin at 37 °C with 5% CO₂ in a humidified atmosphere. To establish erastin-resistant cells, SKOV3 cells were treated with 10 µM erastin for 7 days, OVCA429 cells were treated with 20 µM erastin for 5 days and the remaining cells were washed and cultured in fresh medium. Cell viability was analysed by using the Cell Counting Kit-8 (CCK-8) (Sigma #96992) according to the manufacturer’s instructions. Liproxstatin-1 (Lipo-1), 7-Cl-O-Nec1 (Nec-1s) and Z-VAD-FMK were obtained from Selleckchem; erastin, ferrostatin-1 (Fer-1), N-acetyl cysteine (NAC), desferrioxamine (DFO), Trolox, necrosulfonamide (NSA), sulfasalazine (SAS) RSL3 and FIN56 were purchased from Sigma. CM-H₂DCFDA and C11-BODIPY (581/591) were purchased from Molecular Probes, Invitrogen.

GA was purchased from Sigma Aldrich (USA) and dissolved in PRMI-1640 medium. Necrostatin-1 (Nec-1) was purchased from Sellecachem (USA). Mdivi-1 was procured from Sigma Aldrich (USA). Necrosulfonamide (NSA) was procured from Sigma Aldrich (USA). The antibodies used in the present study included anti-phospho-RIPK1 (Ser166) (Cell Signaling Technology, USA), RIPK1 (Cell Signaling Technology, USA), anti-phospho-RIPK3 (Ser227) (Abcam, USA), RIPK3 (Cell Signaling Technology, USA), anti-phospho-MLKL (Ser358) (Abcam, USA), anti-MLKL (Sigma, USA), anti-PGAM5 (Abcam, USA), anti-phospho-Drp-1 (S637) (Cell Signaling Technology, USA), anti-Drp-1 (Santa Cruz, USA), and anti-GAPDH (Santa Cruz, USA).