Ecb3001d

Osteosarcoma 143B Tk⁻ cells and colorectal cancer HCT116 cells were used, both carrying wild-type mtDNA. 143B cells were purchased from ATCC (#CRL-8303) and HCT116 cells were a kind gift from Prof. Paolo Pinton from the University of Ferrara. Cell origin was authenticated using AMPFISTRIdentifiler kit (Applied Biosystems #4322288) and their STR profile corresponded to their putative background (Supplementary Fig. 1a). For basal conditions, cells were cultivated in Dulbecco’s modified Eagle medium (DMEM) high glucose (Euroclone #ECM0749L), supplemented with 10% FBS (Euroclone #ECS0180L), L-glutamine (2 mM, Euroclone #ECB3000D), penicillin/streptomycin (1 ×, Euroclone #ECB3001D), and uridine (50 µg mL⁻¹, Sigma-Aldrich #U3003), in an incubator with a humidified atmosphere at 5% CO₂ and 37 °C. Cells were replaced by a fresh batch after 15 passages and mycoplasma testing was performed before disposal and after each thawing (approximately every 2 months). Experiments in hypoxia were performed using an Invivo2 300 (Baker Ruskinn) chamber, set at 5% CO₂, 37 °C and 1% O₂. Where indicated, cells were incubated for 3 h with dimethyloxalylglycine [DMOG (1 mM), Sigma-Aldrich #D3695] or MG132 (10 μM, Sigma-Aldrich #M7449) and 1 h with pimonidazole (100 μM, Hypoxiprobe #70132-50-3).

The human ovarian cancer cell lines SKOV3 and OVSAHO were purchased from ATCC^® (Manassas, VA, USA) and JCRB Cell Bank (Japan), respectively. Cells were cultured in High Glucose Dulbecco's Modified Eagle's Medium (DMEM) with sodium pyruvate (Euroclone #ECB7501L) supplemented with 10% FBS South America origin EU Approved (Euroclone #ECS5000L), 2 mM L-Glutamine (Euroclone #ECB3000D), 1% penicillin/streptomycin (Euroclone #ECB3001D) and 50 µg/mL uridine (Sigma–Aldrich #U3003) and maintained at 37 °C in a humidified atmosphere with 5% CO₂. For high- or low-glucose experiments, cells were grown for 24 hours in DMEM (Gibco #11966025) supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin/streptomycin, 50 µg/mL uridine, 1 mM sodium pyruvate (Sigma–Aldrich #P2256) and 25 mM or 5 mM D-(+)-glucose (Sigma Aldrich #G7021). Where indicated, cells were treated with 1 µM N4-[2-(4-Phenoxyphenyl) ethyl]-4,6-quinazolinediamine [EVP-4593; Sigma–Aldrich #SML0579] at multiple timepoints (6 h, 12 h, 24 h) and compared to Time 0 (T0). EVOS M5000 Imaging System (Thermo Fisher Scientific #AMF5000) was used for cell line monitoring.