Establishing Neuroblastoma Cell Line ρ0

The human neuroblastoma cells (SK-N-AS, American Type Culture Collection, Manassas, VA, USA) were cultured with Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), and antibiotic-antimycotic (Invitrogen) in 5% CO₂/95% O₂ humidified incubator at 37 °C. The cells were isolated and then re-cultured at a 1:10 ratio when the cells grew to 60–80% of confluence and maintained in DMEM containing 10% FBS, 25 mM HEPES, 110 mg/mL pyruvate and 50 ng/mL uridine (Invitrogen, Carlsbad, CA, USA) in 5% CO₂/95% O₂ humidified incubator at 37 °C. Furthermore, cells grew in a medium with 50 ng/mL ethidium bromide (EtBr) for 3 months to deplete mtDNA. A single clone was selected by limit dilution. The mtDNA-depleted ρ⁰ cells were confirmed by detection of mtDNA copy number, expression of mtDNA-coded proteins and oxygen consumption in the absence of EtBr [20 (link),21 (link)]. The ρ⁰ cells were subcultured at a 1:3 ratio when the cells grew to 60–80% of confluence.