Pepstatin a

Costunolide (CTL) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and dissolved at a concentration of 100 mM in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-Acetyl-L-cysteine (NAC), 2′,7′-Dichlorofluorescin diacetate (DCFH-DA), Carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and Rhodamine 123 (Rho 123) were purchased from Sigma (St. Louis, MO, USA). Pepstatin A was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Acridine Orange (AO), LysoTracker Red DND-99, and MitoSOX were purchased from Invitrogen (Carlsbad, CA, USA).

Stocks of LY294002, an inhibitor of PI3K-Akt (Sigma-Aldrich), BEZ235, a dual inhibitor of PI3K-Akt and mTOR (Selleck Chemicals), rapamycin, a mTOR inhibitor (Merk, Schiphol-Rijk, The Netherlands) and PF-4708671, an inhibitor of p70S6K (Selleck Chemicals) were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO). All reagents were stored in 25 μL aliquots and frozen at −80°C. Other reagents including Earle's Balanced Salt Solution (EBSS) (Lonza), E-64-d (Santa Cruz Biotech, Santa Cruz, CA) and pepstatin A (Santa Cruz Biotech, Santa Cruz, CA) were also used.

Histopaque, p-nitrophenyl valerate (pNPVa), and LPS (E. coli 055:B5) were purchased from Sigma (St. Louis, MO, USA). AIM V^® Serum Free Medium was purchased from Thermo-Fisher (Waltham, MA, USA). RIPA lysis buffer and protease inhibitors (phenylmethylsulfonyl fluoride, PMSF; 4-(2-aminoethyl)benzenesulfonyl fluoride, AEBSF; bestatin; pepstatin A; leupeptin hemisulfate; and aprotinin) were from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies used for Western blots (anti-CES1, anti-MAGL, β-actin, goat anti-rabbit, and goat anti-mouse) were purchased from Abcam (Cambridge, MA, USA). Authentic 2-AG, anandamide, AA, and its deuterated analog AA-d8 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Small molecules JZL184 and WWL113 were purchased from Sigma. CpG was purchased from InvivoGen (San Diego, CA, USA). Primary CES1 and MAGL antibodies for flow cytometry were purchased from Abcam. Fluorescence secondary antibodies for flow cytometry and antibodies against IL-6 used to neutralize IL-6 or perform ELISA were purchased from Biolegend (San Diego, CA, USA). For some of the experiments, monocyte-depleted (n = 1) and whole PBMCs (n = 5) were purchased from Astarte Biologics (Bothell, WA, USA).

Complete growth medium was replaced by serum-free medium for 24 hours in AP-1 cells cultured on a six-well plate. Conditioned medium was collected, treated with protease inhibitors (pepstatin A, PMSF and E-64, Santa Cruz Biotechnology Inc., Paso Robles, CA, USA), and subjected to zymography. Briefly, conditioned medium was concentrated (Microcon 30 K, Millipore Co, Bedford, MA, USA) and resuspended in SDS-PAGE sample buffer (without β-mercaptoethanol). Protein amount was estimated by BCA assay (Pierce, Rockforf, IL, USA). Samples were separated on 10% polyacrylamide gels containing 0.2% gelatin (Sigma). After electrophoresis, gels were washed in 2.5% Triton X-100 (Sigma) for 30 min and incubated for 16–24 hours at 37°C in a development buffer containing 50 mM Tris pH 8.0, 5 mM CaCl₂ and 0.02% NaN₃. Gels were stained with 0.2% Coomassie blue R250 (Amersham Co Arlington Heights, IL, USA) and destained with acetic acid/methanol. MMP2 and MMP9 activity was observed as clear bands in dark background.