Farnesyl thiosalicylic acid fts

Dulbecco's Modified Eagle Medium (DMEM, low glucose), RPMI1640, DPBS and fluorescein isothiocyanate-dextran molecular weight 70,000 (FDx70) were purchased from Life Technologies. Recombinant mouse macrophage-colony stimulating factor (M-CSF) was from R&D Systems. Phorbol 12-myristate 13-acetate (PMA), U73122 and rottlerin were from Abcam. Calphostin C was from Calbiochem. Farnesyl thiosalicylic acid (FTS) was from Santa Cruz. MK-2206 was from Apexbio.

Anti-TW1 antibody (Ab; ab49254) and ELISA Kits of Ras GTPase and Ras GDPase were purchased from abcam (Cambridge, MA). TW1 CRISPR/Cas9 KO plasmids, Abs of RASAL1 (B-2), STAT3 (F-2), pSTAT3 (23G5), KRas (F234), CD11b (44; AF488), HRP-His tag (H-3), Ly6G Ab (RB6-8C5; AF594), CD45 (35-Z6, AF790), CD11c (3.9, AF680), CD3 (PS1, AF700), CD19 (B-1, AF594), EpCAM (C-10, AF647) and Farnesyl thiosalicylic acid (FTS) were purchased from Santa Cruz Biotech (Santa Cruz, CA). CD66b Ab (AF647) was purchased from BD Bioscience (Franklin Lakes, NJ). PE-Siglec 8 Ab (7C9) was purchased from BioLegend (San Diego, CA). Reagents and materials for immunoprecipitation, RT-qPCR and Western blotting, GRβ (PA3-514), GRα (PA1-516) Abs were purchased from Invitrogen (Carlsbad, CA). Proteins of IL-6 and TGF-β, ELISA kits for determining ECP, NE, MPO, IL-1β, TNF-α, IFN-γ, IL-6 and IL-17 were purchased from R&D Systems (MinneAFolis, MN). Recombinant (r) KRas, His-rRASAL1, rSTAT3, rTW1, rGR and rCortisol were provided by Sangon Biotech (Shanghai, China). Stattic, EDTA, dexamethasone, dextran sulphate sodium, Cobalt chloride, PMA and collagenase IV were purchased from Sigma Aldrich (St. Louis., MO).

Human colon adenoma-carcinoma cell lines HT29, HCT116 and SW480, normal lung epithelial cells Beas2B, and mouse macrophage RAW264.7 were obtained from the American Type Culture Collection (Rockville, MD, USA). These cell lines were cultured in complete Roswell Park Memorial Institute medium (RPMI 1640, Sigma, St. Louis, MO, USA) which was supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). SW480 cells were treated with a sub-lethal dose of farnesylthiosalicylic acid (FTS), an inhibitor of KRAS signaling purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The exosomes isolated from SW480 cells with or without treatment of FTS were used in the study for extracellular vesicle characterization, protein profiling and uptake by different cell types.