Pd098059

Dulbecco Modified Eagle’s Medium (DMEM), RPMI-1640, fetal bovine serum (FBS), penicillin, streptomycin, and phosphate-buffered saline (PBS) were provided from Gibco, Thermo Fisher Scientific (Carlsbad, CA, USA); ECL System and Bradford assay reagents were from Bio-Rad (München, Germany); protease inhibitor cocktail tablets (4693116001) from Roche Diagnostics (Grenoble, France); bovine serum albumin (BSA) (9048-46-8), formaldehyde solution 37% (F15587), methanol (34860), trizma base (1503), glycine (G8898), acrylamide/bis-acrylamide 30% solution (A3699) from Sigma-Aldrich (St. Louis, MI, USA); ProLongTM Gold Antifade Mountant with DAPI (P36935) from Thermo Fisher Scientific; IBAfect reagent (7-2005-050) from IBA Lifesciences (Goettingen, Germany); MitoTracker Red CMXRos (M7512) from Invitrogen (Carlsbad, CA, USA); MEK inhibitor PD098059 [2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one] (10006726) and PI3K inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4- one] (70920) from Cayman Chemical (Ann Arbor, MI, USA).

All the cell lines (RPE1, Hela, U87, HCT116, MDAMB231, HEK 293, Caco-2, HT29, IMR90) were obtained from American Type Culture Collection (ATCC) or Coriell Institute and cultured according to standard mammalian tissue culture protocols and sterile technique. The cell lines were cultured in complete Dulbecco’s Modified Eagle Medium (DMEM) supplemented with Fetal Bovine Serum (FBS) (10%) (Sigma, #F1051) and penicillin-Streptomycin (1%) (Wisent, #450-201-EL). The following reagents were used in cell culture experiments: Tamoxifen (Sigma, #T5648), PD098059 (Cayman Chemical, #10006726), Torin1 (Cayman Chemical, #10997), Cycloheximide (Sigma, #C7698), Puromycin (Sigma, #P8833), Blasticidin S-HCl (ThermoFischer Scientific, #A1113903), Hygromycin B (BioShop Canada, #HYG002), Akt inhibitor VIII (Cayman Chemical #14870), MG132 (Cayman Chemical, #10012628), Doxycycline (Sigma, #D3447), Doxorubicin (Tocris, #2252), Nutlin3a (Cayman Chemical, #18585) and Rapamycin (LC Laboratories, #R5000).

Crude E. cava flake (CEF) was obtained from MilaeML (St. Ogeum, Seoul, Korea). RAW264.7 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Dulbecco’s Modified Eagle’s medium (DMEM), Fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin were purchased from Gibco (Grand Island, NY, USA). LPS (Escherichia coli 0111:B4) was purchased from Sigma (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was purchased from RMS Bio-solution (Seoul, Korea). Thiazolyl Blue tetrazolium bromide (MTT) was purchased from Alfa Aesar (Ward Hill, MA, USA). Griess reagent was purchased from Sigma (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from BioLegend (San Diego, CA, USA). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). PD098059 were purchased from Cayman Chemical (Ann Arbor, MI, USA). Primers were purchased from Bioneer (Daejeon, Korea). Antibodies were purchased from Cell Signaling Technology (Boston, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA).