1 oleoyl lpa

1-Oleoyl LPA (18 : 1 LPA) was purchased from Avanti Polar Lipids (Birmingham, AL, USA). Inhibitor of phosphoinositide 3-kinase (PI3K), LY290042, and inhibitor of mitogen-activated protein kinase (MAPK), PD98059, were from Cell Signaling (Beverly, MA, USA). Rho kinase inhibitor, Y-27632, was from Biomol (Beverly, MA, USA). Hela cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 mL/L fetal bovine serum (FBS), streptomycin (100 mg/L), and penicillin (100 KU/L) at 37°C in 50 mL/L CO₂ incubator. Cells were serum starved for 12 hours before LPA treatment.

Various chemical forms of LPA were assayed: 1-oleoyl-LPA (18:1), 1-palmitoyl-LPA (16:1), 1-arachidonoyl-LPA (20:4), 1-linoleoyl-LPA (18:2), and 1-oleoyl-lysophosphatidylcholine (18:1 LPC) (Avanti Polar Lipids Inc.). Saturated or mono-unsaturated samples (16:0, 18:1 LPAs, and 18:1 LPC) were completely dissolved in ethanol:water (1:1 v/v) by sonicating for 3–5 min, aliquoted in glass vials layered with N₂, and stored under N₂ atmosphere at −20°C for several uses (up to 9 months). Unstable and unsaturated LPA samples (18:2 and 20:4; received in CHCl₃) were desiccated and then reconstituted in fresh ethanol:water (1:1 v/v) for immediate use in binding assays. Redissolving desiccated LPAs in aqueous BSA solutions for storage purposes was eliminated because it resulted in 95–97% loss of LPA during reconstitution (38 (link)). Stored or reconstituted LPAs in ethanol:water solution show a monodispersed distribution of LPA as measured by dynamic light scattering (DLS). Saturated LPAs are relatively stable under atmosphere, whereas unsaturated ones are highly unstable, extremely hygroscopic, and, therefore, cannot be stored for subsequent use in this assay.

Human recombinant ATX [specific activity: 5.40 or 3.13 U/mg (batch number 0530156–1 or 0584094, respectively)] was purchased from Cayman Chemical (Ann Arbor, MI, USA). 1-oleoyl-LPA (LPA 18:1) and LPC 16:0 were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Avanti Polar Lipids (Alabaster, AL, USA). 1-Oleoyl-cPA (cPA 18:1) was purchased from Avanti Polar Lipids. The purity of cPA obtained from Avanti Polar Lipids, which is frequently highly degraded or contaminated with 1-oleoyl-LPA (up to 98%), is usually confirmed using QqTOF. In this study, we used highly pure cPA, purity was confirmed to be above 97%. Additionally, 2ccPA was also prepared as per previously described^{4 (link)}.
3ccPA for TGFα-shedding assay and preparation of the degradation compound, was generously provided by Prof. Susumu Kobayashi (Tokyo University of Science). For other experiments 3ccPA was purchased from Echelon Biosciences (Salt Lake City, UT, USA). N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt, dihydrate (TOOS) was purchased from Dojindo (Kumamoto, Japan). Horseradish peroxidase was purchased from Toyobo Bio (Osaka, Japan). Choline oxidase isolated from Arthrobacter globiformis was kindly provided by Imamura Enzyme Technologies Corp. (Shizuoka, Japan). Acidic methanol (pH 4.0) was prepared as per previously described^{35 (link)}.