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Human OS cell lines U2OS, MG-63, HOS, SJSA-1, and 143B (ATCC, USA) and normal osteoblast cells hFOB1.19 (ATCC, USA) were included in this study. MG-63, HOS, hFOB1.19 and 143B cell lines and SJSA-1 cells were cultured in EMEM (ATCC, USA) with 10% FBS and RPMI-1640 medium (ATCC, USA) with 10% FBS, respectively, at 37 °C (or at 33.5 °C for hFOB1.19 cells) in an incubator with 5% CO₂ and 95% humidity.

The murine cell lines CT26.WT (ATCC CRL-2638) (colon carcinoma, female), B16-F10 (CRL-6475) (malignant melanoma, male) and the human cell line SJSA-1 (CRL-2098) (osteosarcoma, male) were purchased from ATCC. At arrival, the cell lines were expanded and aliquots were frozen at low passage numbers. None of the cell lines were further authenticated. B16-F10 p53^−/−(clone 8) was generated from the B16-F10 cell line,⁴⁰ (link) expanded and frozen.
Prior to experiments, the cell lines were thawed and allowed to adjust to culture for a minimum of one week, until normal growth rate was obtained. All cell lines were grown in RPMI-1640 medium (R8758 Sigma Aldrich) supplemented with 10% heat inactivated FBS (SV30160.03, Hyclone) and 100 U/mL penicillin and 100 μg/mL streptomycin (5140-122, Gibco). The cell lines were sub-cultivated before reaching confluency, at least twice per week. All cell cultures were maintained at 37°C and 5% CO₂. None of the cell lines were maintained in culture for more than five months.
The cultures were tested and confirmed to be negative for mycoplasma infection every second month, using the MycoAlert Plus Detection kit (LT07-710, Lonza) according to the manufacturer’s instructions.

Normal osteoblast cell hFOB1.19 and human OS cell lines, including 143B, SJSA-1, HOS, MG-63, U2OS were purchased from ATCC. All the cell lines were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). All the cells were identified to be mycoplasma-free and were kept at 37°C with 5% CO₂.