2.2. HepG2 Cell Culture and Supplementation

HepG2 human hepatoma cells were maintained at 37 °C, 95% air, 5% CO₂ in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin [19]. Once a week, cells were split 1:20 into a new 75 cm² flask, and the medium was refreshed. Cells were seeded in 12-well plates at 6 × 10⁵ cells/mL concentration, and after 24 h, they were incubated for 6 or 24 h with the bioactive compounds as previously described [20]. DHA was dissolved in 100% ethanol and complexed to bovine serum albumin (BSA). The DHA-BSA complex was prepared fresh each time at a final BSA concentration of 0.5% in serum-free DMEM. PCA was dissolved in dimethyl sulfoxide (DMSO) acidified with HCl to pH 2, while PRO was dissolved in water. The final concentration of ethanol and DMSO was kept below 0.1% in serum-free DMEM. Not supplemented, control cells (Ctrl) received corresponding amounts of BSA, ethanol and DMSO. DHA, PRO and PCA concentrations used in the study (50 μM DHA; 70 μM PRO; 20 μM PCA) were not cytotoxic to HepG2 cells as evidenced by different assays in preliminary experiments [19].