Cell culture and transfection:

Huh7 and HepG2 cells were cultured at 37°C in a humidified incubator at 5% CO₂ in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Different concentrations of FANA ASOs were added directly to the cell culture medium and cells were harvested 48-72hrs post treatment. {"type":"entrez-nucleotide","attrs":{"text":"AC069294.1","term_id":"8051570","term_text":"AC069294.1"}}AC069294.1 expressing plasmid DNA was transfected into Huh7 cells using lipofectamine 2000 (Fisher Scientific, USA), and cells were harvested 72 hrs post transfection. FANA ASOs and transfection experiments were conducted in biological quadruplicates or triplicates. Total RNA was prepared from Huh7 cells using direct-zol RNA miniprep plus kits (Zymo Research, CA, USA). Total protein was prepared from the same cells using acetone precipitation of the flow-through Trizol lysate. Briefly, after passing the lysate through the column, the flow-through was collected, four volumes of acetone were added, the samples were incubated on ice for 30 min, and then centrifuged at 15,000 g for 10 min at 4°C. The pellets were washed once with 400 μl ethanol (95-100%), air-dried, and resuspended in sample loading buffer. Total protein concentrations were measured using the Bradford method (Thermofisher Scientific, CA, USA).