Palmitic acid

The PS container was fabricated by combining 2.0-cm-thick polystyrene boards. A 1.0-cm-diameter hole was created in the lid to direct the generated water vapor and hydrogen outside the container. A 2.0-mm-thick piece of chloroprene rubber was placed between the top of the container and lid, providing a seal between the lid and container. Two bands were placed around the PS container to fix the lid to the container.
The stand and frame were fabricated using a Mojo 3D printer (Stratasys, Rehovot, Israel) with an ABS resin (Mojo P430 QuickPack Print Engine, Stratasys).
The PP container was made of a 200-µm-thick PP film. The stand and 80 g of palmitic acid (Wako Pure Chemical Industries, Ltd., Osaka, Japan) were placed inside the container, and a handle made of PP film was placed on top of the PP container for easy operation. palmitic acid (C₁₆H₃₂O₂) is a type of saturated fatty acid with a melting point of 62.9 °C.
A commercially available exothermic agent (Morians Heat Pack size L; Morian Heat Pack Co. Ltd., Irima City, Japan; http://www.morians.co.jp/morians/structure.html) and a VI container (SR250; ASVEL, Yamatokoriyama-shi, Japan) were also used as components for the device. The main components of the exothermic agent consisted of calcium oxide and aluminum, and 45 g was used in this experiment.

Formic acid, free cholesterol enzymatic test kit, trimethylamine, pyridine, sodium sulphate, palmitic acid, and palladium/carbon were purchased from FUJIFILM WAKO Chemicals (Osaka, Japan). Acetone, chloroform, ethanol, filipin complex, 1-hydroxytriazole, Ham′s F-12 medium, iodine, pivaloyl chloride, serine, and toluene were purchased from Nacalai tesque (Kyoto, Japan). Acetonitrile, methanol-d₄, and chloroform-d were purchased from Kanto Kagaku (Tokyo, Japan). Acetyl chloride, dichloromethane, 1, 3-dicyclohexylcarbodiimide, imidazole, N-methylmorpholine, L-serine benzyl ester hydrochloride, and phosphorus trichloride were purchased from Tokyo chemical industry Co. (Tokyo, Japan). NBD-F was purchased from DONJINDO laboratories (Kumamoto, Japan). Choline tosylate was purchased from Toronto Research Chemicals (North York, ON, Canada). Foetal bovine serum was purchased from Gibco (Grand island, NY, USA). SPC and Lyso-SM (d17:1) were purchased from Avanti polar lipids, Co. Ltd. (Alabaster, AL, USA). Ultrapure water was prepared with PURELAB ultra (Organo Co. Ltd., Tokyo, Japan) and used in all MS analyses.

The Hepa1-6 cell line, which is derived from a BW7756 tumor from a C57BL/6J mouse, was purchased from DS Pharma Biomedical Japan (Osaka, Japan). To observe the effect of steatosis in hepatocytes, Hepa1-6 cells were exposed to 0.1 mM oleic acid or palmitic acid (Wako Chemical, Osaka, Japan) for 3 h, as described previously [21 (link)]. Lipid accumulation was confirmed by Sudan III staining [21 (link)]. The concentrations of CCL2 present in culture supernatants were estimated by ELISAs.

FL, lignoceric acid, palmitic acid, boric acid, potassium chloride, sodium hydroxide, chloroform, methanol, and ethanol were purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Cholesterol, cholesteryl sulfate, octacosanoic acid, 1,6-diphenyl-1,3,5-hexatriene (DPH), N,N,N-trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl)phenyl-ammonium p-toluenesulfonate (TMA-DPH), and 8-anilino-1-naphthalenesulfonic acid ammonium salt (ANS) were from Sigma-Aldrich (St. Louis, MO, USA). Ceramide type III and ceramide type VI were from Evonik Industries AG (Essen, Germany). These reagents were used without further purification.

Bovine brain microvasculature endothelial cells (BBMCs) and human umbilical vein endothelial cells (HUVECs) were used. Cultures were grown for 24 h at 37 °C in a humidified atmosphere of 95% air and 5% CO₂ before use in experiments. BBMCs were plated in a 10-cm dish coated with collagen in containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic-antimycotic (Invitrogen), and 10 ng/mL basic FGF (Roche Applied Science, Penzburg, Germany). HUVECs were also plated in a 10-cm dish coated with collagen in medium (Humedia-EG; Kurabo, Osaka, Japan).
An adenovirus-mediated reporter system was prepared. HUVECs were transfected with purified adenovirus, including miRNAs targeting β-arrestin-2 and control miRNA, and evaluated after 2 days. The miRNA sequence for the knockdown of β-arrestin-2 was 5′-TGCTGATACCTGGTCATCTTGTTCGAGTTTTGGCCACTGACTGACTCGAACAATGACCAGGTAT-3′. The sequence of control miRNA was LacZ-specific miRNAs. HUVECs samples were collected after 24 h in palmitic acid (WAKO) in 100% Ethanol conjugated to 10% bovine serum albumin.