Metabolic Flux Analysis of HepG2 Cells

Metabolic flux analysis was performed as described in ref. 59 (link): Briefly, HepG2 cells were cultured on Seahorse XF-24 (Seahorse BioSciences, Billerica, MA, USA) plates at a density of 7.0 × 10⁴ cells per well. Cells were treated with control vehicle, sesamol (1 mM), rapamycin (100 nM), or sesamol (1 mM) + rapamycin (100 nM). Cells were pre-treated with rapamycin for 30 min and the assays were conducted 24 h post-treatment. On the day of metabolic flux analysis, the media was changed to unbuffered DMEM medium and treated at 37 °C in a non-CO₂ incubator for 1 h. All medium and injection reagents were adjusted to pH 7.4 on the day of assay. Using the Seahorse XF-24 (Seahorse BioSciences) metabolic analyzer, three baseline measurements of OCR and ECAR were sampled prior to sequential injection of mitochondrial inhibitors. Three metabolic determinations were sampled following addition of each mitochondrial inhibitor prior to injection of the subsequent inhibitors. The mitochondrial inhibitors used were oligomycin (4 μM), FCCP (0.2 μM), and rotenone (1 μM). OCR and ECAR were automatically calculated and recorded by the Seahorse X-24 analysis software. After the assays, protein levels were determined by BCA assay for each well to confirm equal cell density per well.