Cell Culture.

HEK293 cells expressing Mito-FAP dL5** (Mito-FAP, FAP cloned downstream of mitochondrial targeting sequence of COXIV/COXVIII and fused with a fluorescent protein mCerulean3 for visualization) (18) were cultured in Dulbecco’s modified Eagle medium (DMEM) media supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin-streptomycin in 5% CO₂ at 37 °C. The HEK293 Mito-FAP ρ⁰ cell line was established by culturing parental HEK293 Mito-FAP cells in DMEM supplemented with 10% heat-inactivated FCS, 1% penicillin-streptomycin, 1 mM sodium pyruvate, 50 μg/mL uridine, and 50 ng/mL ethidium bromide for at least 4 wk (50). pHyPer-nuc plasmids were obtained from Evrogen. Transfection was performed using FuGENE 6 (Roche Diagnostics) according to the manufacturer’s instructions.