Mnng hos

Osteosarcoma cell lines including 143B (CRL‐8303), MG-63 (CRL‐1427), and Saos-2 (HTB‐85) cells were purchased from ATCC (Manassas, VA, USA). U2OS (CLS‐300364) and MNNG/HOS (CLS 300289) were from Cell Lines Service (GmbH, Eppelheim, Germany). All cell lines were cultured and maintained at 37̊C in humidified 5% CO₂ incubator following manufacture's instruction. All osteosarcoma cells were seeded at a density of 20,000 cells per well in a 24 well plate. To determine number of osteosarcoma cells, the cells were trypsinized and counted in hemocytometer counting chamber every 24 h for 7 days using trypan blue exclusion assay. The doubling time could be determined by a cell number along the exponential phase of the growth curve. The doubling time of each cell lines was calculated using the following formula Doubling time =  duration of culture (h)×  ln(2)/ln(c2/c1), where c is the number of cells at each time of collection and ln is a neperian logarithm (Roth V. 2006 Doubling Time Computing, Available from: http://www.doubling-time.com/compute.php)

Osteosarcoma and osteoblastic cell lines used in this study include MNNG/HOS (CLS 300289; Cell Lines Service, GmbH, Eppelheim, Germany), U2OS (CLS-300364; Cell Lines Service, GmbH), 143B (CRL-8303; ATCC, Manassas, VA, USA), MG-63 (CRL-1427; ATCC), Saos-2 (HTB-85; ATCC) and normal human osteoblast cell line hFOB1.19 (CRL-11372; ATCC). MNNG/HOS was cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). MG-63 was cultured in DMEM supplemented with 10% FBS. 143B cells was cultured in DMEM supplemented with 10% FBS and 0.015 mg/ml 5-bromo-2′-deoxyuridine (Merck KGaA). U2OS, Saos-2 and hFOB1.19 were cultured in F-12 Medium supplemented with 10% FBS. All the cells were maintained at 37°C in humidified 5% CO₂ incubator.