9 10 3h oleate

The tritium release assay was performed as previously described, with the noted changes [45 (link)]. Cells were grown in T175 flasks and seeded at 350,000 cells per well in 6-well plates in triplicate and in duplicate wells for protein concentration for normalization, and grown for 24 h in complete DMEM. Wells were treated with REN001 in complete DMEM for 48 h in a 37 °C/5% CO₂ incubator. Cells were washed once with PBS and incubated with 0.34 µCi [9,10-³H] oleate (45.5Ci/mmol; Perkin Elmer, Waltham, MA, USA) in 50 nmol of oleate prepared in 0.5 mL glucose-free DMEM with 1 µg/mL L-carnitine and 2 mg/mL alpha-cyclodextrin for 2 h at 37 °C. Fatty acids were solubilized with alpha-cyclodextrin as described [46 (link)]. After incubation, ³H₂O released was separated from the oleate on a column containing 750 µL of anion exchange resin (AG 1 × 8, acetate, 100–200 Mesh, BioRad) prepared in water. After the incubation medium was passed through the column, the plate was washed with 1 mL of water, which was also transferred to the column, and resin was washed with 1 mL of water. All eluates were collected in a scintillation vial and mixed with 10 mL of scintillation fluid (Eco-lite, MP), followed by counting in a Beckman scintillation counter in the tritium window. Standards contained a 10 µL aliquot of the incubation mix with 3 mL of deionized water and 10 mL of scintillation fluid.