Palmitoleic acid

Human liver hepatocellular carcinoma cell line (HepG2 cells) was
purchased from the American Type Culture Collection (ATCC; Manassas, VA). Cells
were cultured in EMEM (ATCC) supplemented with 10% fetal bovine serum
(Life Technologies; Carlsbad, CA) in 25 cm² polystyrene flasks placed
in a Hera Cell 5% CO₂ 37°C incubator (ThermoFisher
Scientific; Waltham, MA). Routine passage was carried out every 2 or 3 days.
About 2 × 10⁵ HepG2 cells were seeded per 6-cm plate
and serum-starved for 24 h before the following treatments: 50, 100 or 200
μg/ml human LDL-cholesterol (LDL-C) (Kalen Biomedical; Montgomery Vlg,
MD); 10, 30 or 90 μM simvastatin (Sigma; St Louis, MO); 100 nM insulin
(Sigma); 30 nM glucose (Sigma); 5 μM Bay-11 (Cayman; Ann Arbor, MI); 200
μM fatty acids conjugated with 0.2% BSA (30 mM) in 1%
ethanol. We treated HepG2 cells for 24 h with palmitic acid (Sigma), palmitoleic
acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and
eicosapentaenoic acid (Cayman).

Human Umbilical Vascular Endothelial Cells (HUVEC) were purchased from Lonza and were cultured following the vendor’s instructions. To prepare samples, cells were detached from culture dishes using TrypLE Select (Life Technologies) and suspended in growth mediums before being pipetted into the microfluidics devices at cell density of approximately 1800–2000 cells/mm² at 100% confluence (600–800 cells/mm² at 30%−50% confluence in the lower density experiments) allowing the cells to form monolayers. After overnight incubation, fluorescent calcium dye (Calbryte 520, AAT Bioquest) was loaded for 40 minutes prior to imaging. Palmitoleic acid (Sigma-Aldrich, MO) was used as gap junction inhibitor in our experiments. Palmitoleic acid was first dissolved in DMSO and then diluted to 10uM in growth medium. Before experiment, cells were treated with 10uM Palmitoleic acid for 8 to 12 hours while seeding into the PDMS device. Then, 10uM Palmitoleic acid was added to growth medium used for shear-stree experiments.

Azelaic acid, undecanedioic acid, sebacic acid, citric acid, palmitic acid, palmitoleic acid, oleic acid, linoleic acid, phenyl sulfate, taurocholic acid (TCA), and taurodeoxycholic acid (TDCA) were purchased from Sigma-Aldrich (St. Louis, MO). 1-Palmitoyl-2-hydroxy-sn-glycero-3-phospho-ethanolamine (LPE 16:0), 1-stearoyl-2-hydroxy-sn-glycero-3-phos-phoethanolamine (LPE 18:0), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (LPE 18:1), and 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC 18:0) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). Cresol sulfate and cresol glucuronide were synthesized as described previously.^{22 (link)} All solvents and organic reagents were of the highest grade available.

RPMI medium 1640, propidium iodide (PI), ethylenediaminetetraacetic acid (EDTA), β-mercaptoethanol, penicillin and streptomycin, sodium pyruvate, and L-glutamine were purchased from Life Technologies. Fetal bovine serum (FBS), bovine serum albumin (BSA), and phosphate-buffered saline (PBS) were from Sigma-Aldrich. RPMI medium 1640 was supplemented with 10% FBS, penicillin and streptomycin (10 U/ml), sodium pyruvate (1 mM), L-glutamine (2 mM), β-mercaptoethanol (50 μM), and HEPES (100 mM). Purified natural urushiol was a generous gift from Alfred Del Grosso at the Food and Drug Administration (FDA). Synthetic urushiols C15:1, C15:2, and C15:3 were purchased from Phytolab (Germany). Synthetic urushiol C15:0 was obtained from ChromaDex. Palmitoleic acid, squalene, and glyceryl tripalmitoleate were purchased from Sigma-Aldrich. Oleyl palmitoleate and oleyl oleate were from Nu-Chek.