Necroptosis induction in HeLa cells

HEK293T and HeLa were cultured in DMEM/F12 and DMEM high glucose, respectively (Lonza, Switzerland) supplemented with 10% FCS (Life technologies, USA) [13 (link)]. Lipofectamine 2000 (Invitrogen, USA) was used to transfect the different plasmids following the manufacturer’s instructions. HeLa cells were transfected with the different human RIPK1 plasmids (wild type and mutant) together with human RIPK3. After 4 h of transfection, necroptosis was induced by 48-h incubation with 100 ng/mL of human recombinant TNF (Merck-Millipore, USA), 20 μM of z-IETD-FMK caspase-8 inhibitor (BD biosciences, USA), 500 nM of Inhibitor of Apoptosis Protein (IAP) antagonist BV6 (Merck-Millipore, USA), and a SMAC mimetic that blocks cIAP1 and XIAP interaction with initiator caspases and triggers their proteasomal degradation. Necroptosis was blocked by incubating with 1 μM of necrosulfonamide (NSA; Abcam, UK).