Oleic acid

McArdle rat hepatoma cells (McA-RH 7777; LGC Standards AB, Borås, Sweden) were cultured in MEM (PAA Laboratories GmbH, Linz, Austria) containing 20% fetal bovine serum (HyClone Laboratories, South Logan, UT, USA). For assessment of intracellular lipids, the cells were plated in 24-well plates containing glass coverslips and grown in MEM with 2% fetal bovine serum. After 24 hours, the medium was changed to MEM without fetal bovine serum plus 0, 20, or 60 µM oleic acid (Sigma-Aldrich), and after 48 hours, the cells were treated with 1 nM DHT (dissolved in ethanol; Sigma-Aldrich) or vehicle for 24 hours. In separate experiments, the medium was changed to MEM without fetal bovine serum plus 60 µM oleic acid 24 hours after plating, and after 48 hours, the cells were treated with 1 nM DHT, 10 μM enzalutamide (Selleckchem, Houston, TX, USA), or vehicle for 24 hours. Cells were then fixed in 2% formaldehyde for 5 minutes, treated with 20% isopropanol for 0.5 minutes, and stained with Oil Red O for 20 minutes. Next, cells were treated with 20% isopropanol for 0.5 minutes, stained with hematoxylin for 2 minutes, and finally rinsed in tap and distilled water. Cells were photographed with a Zeiss microscope and Oil Red O–stained area per cell was quantified by BioPix iQ 2.1.8 software (BioPix Software).