Hyclone fetal calf serum

The UMR-106 (UMR) cell line corresponds to an osteogenic sarcoma induced in a rat by serial injections of ³²P [40 (link)]. The MNNG/HOS (MN) cell line was derived from an osteosarcoma tumor present in a 13 year-old caucasian female. The cells were then treated with a carcinogenic nitrosamine [41 (link)]. The UMR and the MN cell lines were maintained in Dulbecco’s modified Eagle medium (DMEM, Lonza, Levallois-Perret, France) and DMEM-F12 (Sigma-Aldrich, St Quentin Fallavier, France) respectively, supplemented with 10% Hyclone fetal calf serum (Thermo Scientific, Villebon sur Yvette, France). Populations enriched in CSCs were cultured in ultra-low attachment flasks (Corning, Fisher Scientific, Illkirch, France) in DMEM-F12 methylcellulose 1% supplemented with 10 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL epidermal growth factor (EGF) (Peprotech, Neuilly sur Seine, France) and N2 supplement (Invitrogen, Fisher Scientific). Sphere dissociation for cell dilution was performed each week upon incubation with Accumax (Sigma-Aldrich), a mix of mild proteases, at 37°C for 10 min. The spheres were transferred at least five times before use and were dissociated two days before each experiment in order to obtain small spheres enriched in CSCs.

Mammalian cells were cultured in medium supplemented with 10% HyClone fetal calf serum (FCS; Thermo Scientific) and 2 mM L-glutamine at 37°C under 5% CO₂. HeLa and HeLa M cells were grown in Dulbecco's modified Eagle's medium (DMEM), and hTERT-RPE1 cells were grown in DMEM with Ham's F12 (1∶1) supplemented with 10 µg/ml hygromycin B (Invitrogen). Transient transfection of plasmid DNA was performed using 1 mg/ml linear polyethylenimine (PEI, Sigma-Aldrich) by forming complexes of DNA–PEI at a ratio of 1∶3, and cells were typically assayed 24 h post-transfection. For siRNA transfections, ∼4.5×10⁴ cells were seeded in 3.5-cm dishes for 24 h, transfected with 20 nM siRNA duplexes using INTERFERin (Polyplus Transfection) as specified by the manufacturer, and analyzed 72 h post-transfection. For siRNA transfections, ∼4.5×10⁴ cells were seeded in 3.5-cm dishes 24 h before transfection with 20 nM siRNA duplexes using INTERFERin (Polyplus Transfection) as specified by the manufacturer. Cells were typically assayed 72 h after transfection. For the siRNA rescue experiments, cells were sequentially transfected with GMAP-210 or control siRNA, and 48 h later with DNA encoding siRNA-resistant full-length GMAP-210–Myc or HRD1–Myc. Cells were incubated for additional 24 h, and then they were trypsinised, re-seeded onto coverslips, and assayed after another 24 h.