17 octadecynoic acid 17 odya

Lymphocyte separation medium, aprotinin, dimethyl sulfoxide (DMSO) and solvents for HPLC and LC/MS were purchased from Thermo Fisher Scientific (Ottawa, Ontario, Canada). Dextran, adenosine deaminase, leupeptin and potassium phosphate were obtained from Sigma-Aldrich Canada (Oakville, Ontario, Canada). HBSS was purchased from Wisent Bioproducts (St-Bruno, Quebec, Canada). 19-OH-prostaglandin (PG) B₂, PGB₂, PGB₂-D₄ and 17-octadecynoic acid (17-ODYA) were purchased from Cayman Chemicals (Ann Arbor, Michigan, USA). PF-4708671 was obtained from Abcam (Cambridge, Massachusetts, USA) and LY2584702 from Selleckchem (Houston, Texas, USA). Thapsigargin was obtained from Tocris Bioscience (Ellisville. Missouri, USA). LTB₄ was a generous gift from Dr Louis Flamand (Université Laval, Québec City, Canada). Recombinant CYP4F3A and the NADPH regenerating system were purchased from Corning (Corning, New York, USA). Protease and phosphatase inhibitor cocktail tablets were purchased from Roche (Laval, Quebec, Canada). Primary (anti-phospho-S6 #2211 and anti-S6 #2317) and secondary antibodies were obtained from Cell Signaling (Danvers, Massachusetts, USA). The enhanced chemiluminescent (ECL) substrate was obtained from Millipore Canada Ltd (Toronto, Ontario, Canada).

Nematode growth medium agar with 10 ml 0.1% Tergitol was supplemented with 0.01 mM of EPA as described. Synchronised L2 C.elegans nematodes were grown for 24 h on NGM agar plates with or without 0.01 mM EPA, seeded with E.coli OP50 until they reached L3 stage. Thereafter, the L3 nematodes were carefully harvested and washed three times with sterile M9 buffer. L3 nematodes were transferred to fresh plates and pre-treated for 24 h with 17-octadecynoic acid (17-ODYA) or 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) (Cayman Chemicals) until they reached L4 stage. The compounds were added directly to the bacterial food at a final concentration of 0.05 mM [37 (link), 38 (link)]. In all cases, solvent controls were included. L4 nematodes were infected with C.albicans and 60 nematodes were transferred into 2 ml of liquid medium (80% M9 buffer, 20% BHI, 90 μg/ml kanamycin) in a single well of a six-well tissue culture plate and incubated at 25 °C. Hyphal formation was monitored after 24 h of incubation. In addition, nematodes exposed to EPA and 17-ODYA were monitored for survival by scoring them as either alive or dead. If nematodes did not show any movement in response to mechanical stimulation, they were considered dead and thus removed from liquid medium assay [25 (link)].

Carbaprostacyclin (cPGI₂), AL 8810, baicalein, and 17-octadecynoic acid (17-ODYA) were purchased from Cayman Chemical. 15-Deoxy-Δ^12,14 PGJ₂, PGE₂, PGF_2α, SC-560, SC-236, and indomethacin were all purchased from Sigma-Aldrich. PD98059, U0126, and GF109203X were obtained from Enzo Life Sciences and BAPTA-AM from Tocris Bioscience.