Stearic acid

Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Hyclone (Logan, UT, USA). Phosphate-buffered saline, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco-BRL (Gaithersburg, MD, USA). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies Inc. (Rockville, MD, USA). The bicinchoninic acid assay (BCA) kit, IFN-γ and TNF-α were purchased from Thermo Fisher Scientific (Waltham, MA, USA). L-leucine, isoleucine, phenylalanine, tryptophan, linolenic acid, palmitic acid and stearic acid were purchased from Cayman chemical (Ann Arbor, MI, USA). The 15-hydroxy-5,8,11,13-eicosatetraenoic acid, stearidonic acid, eicosapentaenoic acid, arachidonic acid, and linoleic acid were obtained from Targetmol (Boston, MA, USA). MS-grade acetonitrile, water and formic acid were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The p-STAT1, STAT1, p-IκBα, IκBα, p-p38, p38, p-ERK, ERK, p-JNK, JNK, p65, and β-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). p50 and anti-proliferating cell nuclear antigen antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cells were seeded in 96-well plates (Corning 3917, 3125–6250 cells per well) and treated with compounds 24 h after plating. Compounds were purchased from Cayman Chemical with the exception of stearic acid, oleic acid, adrenic acid, linolenic acid, linoleic acid, docosahexaenoic acid, conjugated linoleic acid (16413), bovinic acid, arachidonic acid, Z-VAD-FML, and (+)-α-tocopherol (T3634), which were purchased from Sigma Aldrich. RSL3 was purchased from Selleckchem, catalpic acid, α-calendic acid, β-calendic acid, and β-ESA were obtained from Larodan Fine Chemicals, and punicic acid was purchased from Matreya LLC. Initial aliquots of ferrostatin-1, deferoxamine, and ML162 were generous gifts of the Dixon Lab (Stanford) and additional quantities were purchased from Cayman Chemical. The inhibitor of FSP1^{9 (link)} was purchased from ChemBridge Corp. Staurosporine was purchased from LC Sciences. Cell viability was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer’s instructions. Luminescence was measured on an EnSpire Alpha (Perkin Elmer) using the integrated software package. Data were normalized to vehicle-treated or sensitizing agent-alone controls and IC₅₀ curves were produced with GraphPad Prism (Version 8).

Six-well NGM dishes were prepared with an aqueous solution of NP-40 (Sigma) to a final concentration of 0.01% and supplemented with 1 mM concentrations of any of the following reagents: stearic acid (Cayman Chemicals), oleic acid (Cayman Chemicals), linoleic acid (Cayman Chemicals), α-linoleic acid (Cayman Chemicals), γ-linoleic acid (Cayman Chemicals), stearidonic acid (Cayman Chemicals), dihomo-γ-linoleic acid (Cayman Chemicals), eicosatetraenoic acid (Cayman Chemicals), ω-3-arachidonic acid (Cayman Chemicals), arachidonic acid (Cayman Chemicals), eicosapentaenoic acid (Cayman Chemicals), C15iso (Cayman Chemicals), C17iso (Cayman Chemicals), and C13iso (Chem Cruz). Dishes without lipids were also prepared. A synchronized population of 500 L1 animals was added to the wells and incubated for 36 h. Then the animals were challenged with Orsay virus for 3 days and collected for RNA extraction and qRT-PCR. Three independent experiments in triplicate were performed.