3h oleic acid

Mice (12 h fasted, n = 6–7/genotype) were injected retro-orbitally with 100 µL PBS containing 2 µCi of [³H]oleic acid (Perkin Elmer) and perfused with 10 mL PBS through the left ventricle 5 min after injection. Excised tissues were homogenized in 500 µL PBS and aliquots (100 µL) added to 5 mL scintillation fluid (Optiphase HiSafe 3, Perkin Elmer) for radioactivity measurement. Tissue FA uptake was adjusted by tissue weight and by antrum radioactivity which did not differ between groups.

For analyzing LDL‐induced cholesterol esterification, degron‐ORP2 and control cells were cultured in 5% LPDS for 24 h, washed with PBS, and treated with 50 μg/ml LDL for 0–6 h. Cells were then supplemented for 2 h with [³H]oleic acid (5 μCi/ml, Perkin Elmer) in 2% defatted BSA prepared in 5% LPDS medium. During LDL and oleic acid loading, the degron‐ORP2 cells were supplemented with 100 μg/ml IAA as indicated. The cells were collected by scraping in cold 2% NaCl and lipids were extracted as described (Bligh & Dyer, 1959 (link)). Dried lipids were dissolved in chloroform/methanol (9:1), resolved by TLC using hexane/diethyl ether/acetic acid (80:20:1) as the mobile phase and dried plates were stained with iodine vapor. The cholesteryl ester bands were scraped and radioactivity measured by liquid scintillation counting. The results were corrected for the volume and procedural losses and plotted against the total amount of protein in the sample.

ACAT activity was performed as previously described [39 (link)]. In brief, Huh7 cells were seeded in 24-well plates in MEM supplemented with 10% FCS (both from Euroclone, Milan, Italy) at a density of 30,000 cells/well and allowed to adhere for 24 h. After gene silencing previously described, cholesterol esterification was measured by incubating cells with [1-¹⁴C]oleic acid (0.85 µCi/sample; Perkin Elmer, Waltham, MA, USA) complexed with fatty acid-free bovine serum albumin (BSA; Merck, Darmstadt, Germany) for 4 h. At the end of the incubation period, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and a fixed amount of [³H]oleic acid (0.005 µCi/sample; Perkin Elmer, Waltham, MA, USA) was added to each sample as an internal standard. Cellular lipids were extracted by incubating monolayers with a mixture of hexane/isopropanol (3:2) for 30 min with gentle shaking. The extracted lipids were separated by thin layer chromatography (TLC) using a mixture of isooctane/diethyl ether/acetic acid (75:25:2, v/v/v) as mobile phase. Esterified cholesterol radioactivity in each spot was quantified by liquid scintillation counting (Perkin Elmer, Waltham, MA, USA). Data were expressed as cpm of [1-¹⁴C]oleic acid corrected per microgram of protein of each cell lysate measured by the BCA assay according to the manufacturer’s instructions.

U2OS cells were treated with NMIIa or control siRNA for 48 h. Cells were washed 3 × with PBS and then treated with ³H-oleic acid (Perkin Elmer) in DMEM plus 2% fatty acid-free bovine serum albumin for 2 h at 37 °C. Cells were washed 3 × with PBS, lipids were extracted and neutral lipids resolved by TLC. TGs were scraped into scintillation solution and radioactivity was measured. Counts were normalized to an internal standard and protein content. For fatty-acid uptake, experiments cells were treated with ³H-oleic acid in U2OS growth medium for 15 min. Cells were washed 3 × with PBS and cell extracts were made in 2% NaCl. Radioactivity of the cell lysate was measured with scintillation counting and normalized to protein content.

To analyze cholesterol esterification, MEFs were first cultured in 5% LPDS for 72 h, washed with PBS and labeled with [³H]oleic acid (5 μCi/ml, Perkin Elmer) in serum-free, 2% defatted BSA (Sigma) medium for 6 h. During labeling, the cells were supplemented with 50 μg/ml LDL, 2 µM U18666A (Sigma) or 2 µM PKF (i.e., Sandoz 58-035, Sigma) as indicated. The cells were collected by scraping in cold 2% NaCl and lipids were extracted by the Blight and Dyer method^{38 (link)}. Dried lipids were dissolved in chloroform/methanol (9:1), resolved by TLC using hexane/diethyl ether/acetic acid (80:20:1) as the mobile phase and visualized by charring. The cholesteryl ester bands were scraped and radioactivity measured by liquid scintillation counting. The results were corrected for the volume and procedural losses and plotted against the total amount of protein in the sample.