9 10 3h n oleic acid

Fatty acid uptake and incorporation into lipids as well as de novo lipogenesis were determined using [³H-oleic acid and [¹⁴C]-acetic acid, respectively, following the procedure adapted from our previous study (22 (link)). Day 7 differentiated Abhd4 KO and WT 3T3-L1 adipocytes were labeled with 0.5 μCi of [1,2–¹⁴C]-acetic acid (PerkinElmer) or 5 μCi of [9,10-³H(N)]-oleic acid (PerkinElmer) plus 0.04 mM oleic acid (Sigma-Aldrich) conjugated with 0.01 mM fatty acid free-bovine serum albumin (BSA) per ml of DMEM supplemented with 10% FBS, 1% P/S and 100 nM insulin for 0 (no radioisotopes), 30, 60 and 120 min. Following radiolabeling, cells were washed with ice-cold DPBS twice and lipid-extracted with hexane:isopropanol (3:2, vol:vol). Lipid classes from standards and cellular lipid extracts were separated by thin layer chromatography using Silica Gel plates and a solvent system containing hexane:diethyl ether:acetic acid (80:20:2, vol:vol:vol). Lipids were visualized by exposure to iodine vapor, and bands corresponding to TAG, free cholesterol (FC), cholesteryl ester (CE), and phospholipid (PL) were scraped and counted using a scintillation counter. After lipid extraction, cell residue was dissolved with 0.1 N of NaOH, and protein concentrations were measured using a Pierce™ BCA Protein Assay Kit for protein normalization of data.

E. coli LPS (serotype 0127:B8), Leibovitz L15 medium, oligomycin, FCCP (carbonyl cyanide-p- trifluoromethoxyphenylhydrazone), rotenone, antimycin, myxothiazol, α-tocopherol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,7-dichlorofluorescein-diacetate (DCF-DA), and lipid standards were from Sigma-Aldrich (St. Louis, MO, USA). Nile Red was from Invitrogen (Carlsbad, CA, USA). [9, 10- ³H(N)]-oleic acid was from Perkin Elmer (Boston, MA, USA). 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1) was purchased from Molecular Probes (Eugene, OR, USA). MitoQ was a kind gift from Dr. Michael Murphy (Cambridge, UK).