Quantitative real-time PCR

HepG2 cells (5 × 10⁴) were seeded in 24-well plates and, after reaching approximately 70% confluence, were treated in the presence or absence of OA, alone or in combination with EAF at 100 or 200 µg/mL. After incubation for 18 h, total RNA was isolated by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Real-time RT-PCR was performed with an I Cycler iQ (Bio-Rad, Hercules, CA, USA) using SYBR Green PCR master mix (Thermo Fisher Scientific, Waltham, MA, USA). PCR amplification was carried out with the following primers: for sterol regulatory element-binding protein 1 (SREBP-1c), forward 5′-AAACTCAAGCAGGAGAACCTAAGTCT-3′, reverse 5′-GTCAGTG TGTCCTCCACCTCAGT-3′; for ATP citrate lyase (ACLY), forward 5′-TACCACCTCAGCCATCCAGA-3′, reverse 5′-GACCCCAACGAGACCAAGTT-3′; for fatty acid synthase (FASN), forward 5′-AACCGGCTCTCCTTCTTCTTCGACTT-3′, reverse 5′-TCCGAGCGGCAGTACCCATTC-3′; and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward 5′-ATGTTCGTCATGGGTGTGAAC-3′, reverse 5′-GCATGGACTGTGGTCATGAGT-3′.

All mRNA expression was normalized by using GAPDH. Reactions were performed in triplicate, and relative expression levels and standard deviation (SD) values were calculated by applying the comparative method.