Gsk 872

Cells were pretreated with recombinant mouse interferon alpha (IFNα, Calbiochem) at 100 U/mL (L929) or 1000 U/mL (MEF and primary MEF) for 18 hours before being treated with sodium arsenite at 500 μM (L929) or 50 μM (MEF and primary MEF) unless stated otherwise. To induce the canonical necroptosis pathway, cells were pretreated with carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK, pan-caspase inhibitor, ApexBio) at 100 μM, one hour before being treated with 20 ng/mL mouse tumor necrosis factor alpha (TNF-α, Sigma), and continuously treated thereafter. To determine the requirement for the activity of the RIP proteins, cells were pretreated with N-(6-(isopropylsulfonyl)quinolin-4-yl)benzo[d]thiazol-5-amine (GSK872, RIPK3 inhibitor; GlaxoSmithKline) at 3 μM and 2,2-dimethyl-1-(5(S)-phenyl-4,5-dihydro-pyrazol1-yl)-propan-1-one (GSK963, RIPK1 inhibitor; GlaxoSmithKline) at 5 μM, one hour before being treated with sodium arsenite in the presence of the RIPK1 or RIPK3 inhibitor. To inhibit canonical stress granule formation, cells were pretreated with cycloheximide (CHX) (Sigma) at 20 μg/mL for 2 hours or with integrated stress response inhibitor (ISRIB) (Sigma) at 1 μM overnight (14 to 16 hours) before being treated with sodium arsenite.

The following chemical reagents were used in cell culture experiments: LPS-EB Ultrapure (1 μg/ml, Invivogen), zVAD (50 μm, SM Biochemicals), GSK 963 (100nM, GlaxoSmithKline), GSK 843 (100nM, GlaxoSmithKline), GSK 872 (100nM, GlaxoSmithKline), AP1 (100 nM, ClonTech, sold as “B/B Homodimerizer”), itaconate (1mM, Sigma), citrate (1mM, Sigma), dimethyl malonate (1mM, Sigma), 4-octyl itaconate (1mM, provided by Luke O’Neill, Trinity College, Dublin). For in vivo intracranial injections, the following concentrations were used: GSK 963 and GSK 843, 800nM; dimethyl malonate and 4-octyl itaconate, 8mM.

The following PRR ligands and chemical inhibitors were used in cell culture experiments: Poly(I:C) (1 μg/ml, Millipore), LPS-EB Ultrapure (1 μg/ml, Invivogen), CL264 (1 μg/ml, Invivogen), zVAD (50 μM, SM Biochemicals), necrostatin-1 (30 μM, Sigma), GSK 963 (100nM, GlaxoSmithKline), GSK 843 (100nM, GlaxoSmithKline) (Mandal et al., 2014 (link)), GSK 872 (100nM, GlaxoSmithKline) and QVD (20 μM, SM Biochemicals), AP1 (100 nM, ClonTech, sold as “BB Homodimerizer”).