Pf 4708671

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).

Human mesothelioma cells, NCI‐H28, NCI‐H226, MSTO‐211H and NCI‐H2452, and immortalized cells of mesothelium origin, Met‐5A, were purchased from American Type Culture Collection (Manassas, VA, USA). Mesothelioma with mutated p53 genotype, EHMES‐1 and JMN‐1B cells were provided by Dr. Hironobu Hamada (Hiroshima University, Japan) (Nakataki et al., 2006). PEM‐resistant H28‐PEM, H226‐PEM, 211H‐PEM, and H2452‐PEM cells were previously established from the respective parent cells by a stepwise increase of PEM. CDDP‐resistant NCI‐H28, MSTO‐211H and NCI‐H2452 cells were also established with the same method (Kitazono‐Saitoh et al., 2012). Cells were cultured with RPMI‐1640 medium supplemented with 10% fetal calf serum and confirmed to be negative for mycoplasma. The genotype of p53 was wild‐type in NCI‐H28, NCI‐H226, MSTO‐211H and NCI‐H2452 cells, but p53 protein of NCI‐H2452 cells was truncated (Di Marzo et al., 2014). Chemicals used in the present study were purchased as follows: PEM (Eli Lilly, Indianapolis, IN, USA), A769662 (Catalogue number: ab120335), PF4708671 (ab141993), compound C (ab120843, Abcam, Cambridge, UK), nutlin‐3a (S8059, Selleck, Houston, TX, USA), MK‐2206 (CT‐MK2206, ChemieTek, Indianapolis, IN, USA) and rapamycin (R8781, Sigma‐Aldrich, St. Louis, MO, USA).