P5585

Human umbilical vein endothelial cells (HUVECs) were obtained from Procell (CL-0122) and cultured in Dulbecco's modified Eagle's medium (DMEM, HyClone) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). Cells from passages 3 to 8 were used for experiments. Palmitic acid (PA; P5585, Sigma), a representative FFA in vivo, was prepared in dimethyl sulfoxide (DMSO). Confluent HUVECs were serum-starved for overnight in non-FBS DMEM, and then the medium was replaced with FBS-containing DMEM. HUVECs were incubated with PA and/or recombinant human omentin-1 (9137-IN-050, R&D) as follows: control (0 mM PA in phosphate-buffered saline [PBS]), DMSO (0 mM PA in DMSO), 1 mM PA, 1 mM PA + 100 ng/mL omentin-1, 1 mM PA + 150 ng/mL omentin-1, or 1 mM PA + 200 ng/mL omentin-1 for 24 h.
THP-1 cells were purchased from Procell (CL-0233) and cultured in Roswell Park Memorial Institute (RPMI)-1640 (HyClone) medium supplemented with 15% FBS and 1% P/S. Cells from passages 3 to 5 were used for experiments.

Huh-7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, in a humidified atmosphere containing 5% CO₂ at 37 °C. For experimental purposes, cells (2–3 × 10⁶) were seeded in 100 mm culture dishes and, after 24 h, the culture medium was carefully removed and cells were washed twice with phosphate-buffered saline (PBS). Then, cells were treated for 16 h with 400 µM fatty acids (PA or OA), or Tg (2.5 nM), or SCD1i (5 µM) or vehicle (0.2% ethanol + 0.01% DMSO). Palmitic acid (P5585, Sigma-Aldrich) and oleic acid (O1008, Sigma-Aldrich) were dissolved in ethanol at a stock concentration of 148 mM; final concentration of ethanol was 0.2% in the medium. Tg (T9033, Sigma-Aldrich) and SCD1i (sc-205109A, Santa Cruz Biotechnology) were dissolved in DMSO at a stock solution of 2 mM and 25 mM, respectively; final concentration of DMSO was ≤ 0.01% in the medium. All treatments were carried out in serum-free medium containing 0.1% free fatty acids-Bovine Serum Albumin. Media were collected to isolate EVs by a differential centrifugation^{26 (link),45} (see below). Cells were recovered and counted by Countess TC20 automated cell counter (Bio-Rad). Cell viability was assessed by Trypan Blue stain exclusion.