Hos cells

The highly aggressive murine OS cell line LM8 was obtained from RIKEN BRC (Ibaraki, Japan). NIH/3T3 cells, HOS cells (non-aggressive, wild-type k-Ras, human OS), and 143B cells (highly aggressive; k-Ras activated, human OS) (20 (link), 21 (link)) were obtained from the ATCC (Manassas, VA, USA). These cells were cultured in Advanced DMEM (Thermo Fisher Scientific, Waltham, MA, USA) with 2% heat-inactivated FBS (Bio-West, Nuaillé, France). LM8-WT or LM8 TSG101-KO cells were generated by the CRISPR–Cas9 system. The pX330Puro or pX330Puro-TSG101 plasmid was transfected into LM8 cells using ViaFect (Promega, Madison, WI, USA). LM8 TSG101-KO cells were monocloned, resulting in the generation of two monoclonal LM8 TSG101-KO cell lines (KO1 and KO2). LM8-WT cells were maintained as a polyclonal population.

HOS cells were purchased from ATCC (USA). Dulbecco’s modified Eagle medium (Gibco, USA), supplemented with 10% fetal bovine serum (Wisent, Quebec, Canada) and 100 μg/mL pen/strep (Beyotime Biotech, Shanghai, China) was used to culture HOS cells at 37°C with 5% CO₂.