Quantifying Mitochondrial and Nuclear DNA Damage

We assessed nuclear and mtDNA damage as described previously [8 (link),12 (link),31 (link)]. Genomic DNA from cultured cells or paraffin-embedded lungs were extracted using the Qiagen Genomic Tip 20/G and Qiagen DNA Buffer set (Qiagen, Gaithersburg, MD, USA) according to the manufacturer’s protocol. Ex-Taq (Takara, Mountain View, CA, USA) was used for PCR, with specific primers to amplify a mitochondrial genomic in both short and long-form nuclear DNA (beta-globin) [8 (link),12 (link)]. For DNA quantification, we used PicoGreen (Thermo-Fisher/Invitrogen, Waltham, MA, USA) using the FL600 microplate fluorescence reader, with excitation and emission wavelengths of 485 and 530 nm, respectively. Mitochondria small fragment data were used to normalize fluorescence from the mitochondria long fragment. The number of mitochondrial lesions was calculated by the equation: