Dup 697

Human aorta endothelial cells line (HAEC) was obtained from Lonza (Basel, Switzerland). Vascular smooth muscle cell line (MOVAS) was purchased from American Type Culture Collection (ATCC, Rockville, Maryland, MD, USA). HAECs were maintained in supplemented endothelial growth medium EGM-2 (Lonza, Basel, Switzerland), MOVAS cells were cultured in DMEM supplemented 10% (v/v) FBS (both from Gibco, Scotland, UK). Cultured were maintained at 37 °C in a humidified atmosphere of 5% CO₂—95% air. When cells achieved 90% confluence the medium was changed and cells were treated with 100 μM deuterated Oleic acid (OAd34, Sigma Aldrich) or deuterated arachidonic acid (AAd8, 25 µM, Cayman Chemical) in the presence or absence of lipolysis inhibitor, atglistatin (50 µM), phospholipase A₂ inhibitor (AACOCF3, 10 µM, Abcam), COX-1 inhibitor (SC-560, Cayman Chemical) or COX-2 inhibitor (Dup-697, Cayman Chemical) for 4 h or 24 h. Oleic acid (Cayman Chemical) for the experiments was freshly saponified using 100 mM NaOH and BSA-conjugated (10% in DPBS, low endotoxin, fatty acid-free, suitable for cell culture, sterile-filtered; Sigma Aldrich). After 4 h or 24 h of incubation cells were fixed for 4 min with 4% paraformaldehyde. Fixed cells were washed 3 times with DPBS and stored in DPBS at 4 °C until the execution of the measurements.

COX1 and COX2 inhibition assay used the COX Activity Assay kit (cat # 760151, Cayman Chemicals, Ann Arbor, MI, USA) to investigate the OC treatments effect on the enzymatic activity of COX enzymes versus the standard COX inhibitors DuP-697 (Cayman Chemicals, cat # 70645) and SC-560 (Cayman Chemicals, cat # 70340). DuP-697 is a selective COX2 diaryl heterocyclic inhibitor while the SC-560 is a selective COX-1 inhibitor. Cells lysate samples were processed and each sample was assessed run as described by Cayman Chemicals. The colorimetric COX assay was measured by monitoring the appearance of colorimetric oxidized N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) at 590 nm, which was translated to COX activity as per the manufacturer’s protocol.

The test compound was diluted to 10X concentration in 10% DMSO in assay buffer and 10 μL of the dilution was added to a 100 μL reaction so that the final concentration of DMSO from the compounds is 1% in all reactions. All reactions were conducted at room temperature. The reaction mixture contained 60 μL of 1X COX Assay Buffer (BPS Bioscience), 10 μL of enzyme (360 ng COX1 and 250 ng COX2 per reaction) (BPS Bioscience), 10 μL of test compound, 10 μL of 1 mM Ampliflu Red (Sigma-Aldrich), and 10 μL of 0.5 mM arachidonic acid (Cayman Chemical). SC-560 and DuP-697 (Cayman Chemical) were used as reference compounds for inhibiting COX-1 and COX-2, respectively.