Mitochondrial DNA quantification

A day after A549 cells were seeded on 6-well plate (Corning), CSG (200 nM) was treated for indicated times under 5% CO₂ at 37 °C. The media was aspirated, then the cells were washed with PBS. Cells were collected, and genomic DNA was prepared with DNeasy DNA extraction kit (Qiagen) according to the manufacturer’s instructions. The DNA was quantified using NanoVue (Cytiva). 20 ng of genomic DNA was subjected to quantitative PCR (qPCR), combined with 1× KAPA SYBR FAST ABI Prism qPCR master mix (KAPA Biosystems), and forward/reverse primers (200 nM) against human mitochondrial DNA (forward: 5′-CCCCACAAACCCCATTACTAAACCC A-3′; reverse: 5′-TTTCATCATGCGGAGATGTTGGATGG-3′) or human β-actin (forward: 5′-TGTGTGGGGAGCTGTCACAT-3′; reverse: 5′-CGCCTAGAAGCATTTGCGGT-3′) for a final volume 20 μL with nuclease-free water. The PCR was conducted with StepOnePlus (Applied Biosystems), and the PCR cycling was used; initial denaturing at 95 °C for 3 min, followed by 40 cycles of denaturing at 95 °C for 3 s, and extension at 60 °C for 25 s. The data were analyzed by the comparative C_t method, and the mitochondrial DNA levels were normalized to β-actin levels.