Bovine calf serum (bcs)

An aqueous stock solution (60 mM) of sodium bathocuproine disulfonate (BCS, Acros Organics) was prepared and calibrated by competition titration against MCL-1 as previously described.^{16 (link)} BCS (1.50 mM), sodium ascorbate (150 μM) and CuSO₄ (30 μM) were sequentially added via aqueous stock solutions to 3 mL of buffer (10 mM PIPES, 0.1 M KCl, pH 7.0) in a 1-cm path length cuvette equipped with magnetic stirring, and a UV-Vis absorption spectrum was recorded from 650–380 nm. PhenPS was added in 5 μM aliquots from a 2.88 mM stock solution in DMF and an absorption spectrum was acquired after a 2 min equilibration period following each aliquot. The titration was conducted in duplicate, and each dataset was analyzed by nonlinear least-squares fitting over the spectral range from 650–400 nm using the Specfit software package.¹⁷ The stability constant for naphPS was determined similarly, except that the ligand was delivered from a 3.0 mM stock solution in DMSO, and a 5 minute equilibration period was required to obtain a stable spectrum after each aliquot. The phenPS stability constant was further verified by direct competition with MCL-1 as previously described for PSP-2 (Figure S9, Supporting Information).^{10 (link)}

Metrizamide was purchased from Amresco (Solon, OH). Vinblastine sulfate, sucrose, gentamycin, Trizma-hydrochloric acid, potassium chloride, sodium chloride, d-glucose, calcium chloride, protease inhibitor cocktail (for mammalian cell and tissue extracts, in DMSO solution), sodium hydroxide, and magnesium chloride were from Sigma Aldrich (St. Louis, MO). Glycyl-L-phenylnaphthylamine was from Bachem (Torrance, CA). Methanol was from Fisher Scientific (Fairlawn, NJ). Dichloromethane was from Mallinkrodt (Phillipsburg, NJ). Ultra LC/MS-grade Methanol, acetonitrile, and water were from JT Baker (Center Valley, PA). Formic acid was from EMD (Darmstadt, Germany). Penicillin-streptomycin solution (PS) was from Life Technologies (Grand Island, NY). Anti-TNP IgE antibody was from BD Biosciences (San Jose, CA). CXCL10 was from Shenandoah Biotechnology (Warwick, PA). Dubelco’s Modified Eagle Medium (DMEM) high glucose cell medium, bovine calf serum, and fetal bovine serum were from Thermo Scientific (Waltham, MA). Standards including: 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-hexadecanoyl-sn-glycero-3-phosphoethanolamine, and D-erythro-sphingosine (C17 base) were purchased from AvantiPolarLipids (Alabaster, AL).

Caenorhabditis elegans were cultured on nematode growth medium plates spotted with OP50 bacteria and maintained at 15°C or room temperature (approximately 22°C). N2 was used as the wild-type control strain [49 ]. Some strains were provided by the Caenorhabditis Genetics Center, funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). Only healthy animals that were not used in previous procedures and were naïve to testing were used. See the Key Resources Table for strain list.
Low-passage NIH3T3 fibroblasts (authenticated by ATCC and of unknown sex) were cultured in DMEM with 10% bovine calf serum (Thermo Fischer). For polarization assays, cells were serum starved for 2 days, wounded and stimulated with 10 μM LPA (Avanti Polar Lipids, Inc.) [24 (link), 50 (link)]. Cells were grown at 37°C in 5% CO₂.