mtDNA copy number assay

The HepG2 cells were seeded into a 24-well plate at a density of 2 × 10⁵ cells/well. After 24 h, these cells were treated with 1 nM–1 µM of tamoxifen and left in an incubator for 12 days (37°C, 5% CO₂). The reagents and media were replaced every 4 days after washing the cells with phosphate-buffered saline (PBS). To extract the total DNA from the cells and peripheral blood using the total DNA extraction kit (BIONICS, Cat. No. DN40200), the DNA concentration was made up to 50 ng/µl after isolating the total DNA. To measure the mtDNA copy number, the TaKaRa Set (no. 7246, Takara Bio USA, Mountain View, CA) was used to perform real-time PCR (Roche LC480). The mtDNA copy number was obtained using the following formula: 2^{(nDNAprimerCtValue − mtDNAprimerCtValue)}.