Crl 8303

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).

MG63 cells (CRL-1427™-ATCC, Human osteosarcoma) and 143B cells (CRL-8303™- ATCC, Human osteosarcoma) were cultured with Eagle’s minimum essential medium (Gibco BRL, Rockville, MD, USA). The medium contained 10% FBS (fetal bovine serum) and glutamine–penicillin–streptomycin (2 mM–00 U/mL–100 µg/mL) (Gibco BRL). Cells were incubated under a humidified atmosphere of 5% CO₂ room air at 37 °C. For subculture, the cells were treated with trypsin-EDTA (Gibco BRL). After centrifugation of the cells and removal of the supernatant, the cells were replanted into the dish. When the connected cells reached confluence, they had the shape of cobblestones under a microscope.

The murine osteosarcoma MOS-J cell line, called MOS-J/Native in this study, derived from a spontaneous C57BL/6 mouse osteosarcoma, was provided by Prof. L. Shultz [21 ]. Two subclones derived from this cell line were also used in the experiments [6 (link)]. The first clone, MOS-J/PG1, revealed a high proliferation rate in vitro in contrast to the second, MOS-J/A3N. These clones, as well as the MOS-J/Native, were grown in RPMI1640 medium (Lonza, Walkersville, MD, USA) supplemented with 5% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and a mix of 100 U/mL of penicillin and 100 µg/mL of streptomycin (Lonza). The human osteosarcoma cell lines G292 (clone A141B1), KHOS/NP (R-970-5) named HOS in the manuscript, 143B, MG63, SJSA-1, and SaOS2 were purchased at the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively, under references CRL-1423, CRL-1554, CRL-8303, CRL-1427, CRL-2098, and HTB-85. The CAL-72 cell line was purchased at the DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ, Leibniz Institute, Braunschweig, Germany) under reference ACC-439. G292, HOS, 143B, MG63, SaOS2, and CAL-72 cell lines were cultured in DMEM (Lonza) and the SJSA-1 cell line in RPMI-1640 (Lonza), both supplemented with 10% FBS.

The human osteosarcoma cell line 143B was obtained from the American Type Culture Collection (ATCC® CRL8303™). Cell line 531MII was developed at the University Clinic of Navarra and corresponds to a metastatic bone implant from an adult patient with a metastatic bone implant. The clinical characteristics and also the molecular hallmarks (RB1 LOH and TP53 mutation) of the cell line have been reported previously by our group [16 (link)]. Cells were cultured in α-Minimum Essential Medium (a-MEM) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO₂ at 37 °C.

The osteosarcoma cell line (143B, ATCC: CRL-8303, and HOS, ATCC: HTB-96TM, were purchased from the American Type Culture Collection) were cultured in DMEM/F-12 medium (DMEM, Gibco, Shanghai, China) containing 10% fetal bovine serum (Gibco, American), and 100 µg/mL streptomycin and 100 U/mL penicillin (Solarbio, Beijing, China). All the cells were placed in an incubator containing humidified air with 5% carbon dioxide at 37°C, and the medium was replaced with fresh medium once every 3 days.