Ultra low attachment surface 6 well culture plates

The U251 glioma stem cell (GSC) cells were cultured in serum-free DMEM supplemented with B-27 (Gibco; Thermo Fisher Scientific, Inc., NY, USA), 20 ng/ml basic fibroblast growth factor (bFGF; Sigma-Aldrich; Merck KGaA, St. Louis, MO, USA) and 20 ng/ml epidermal growth factor (EGF; Sigma-Aldrich; Merck KGaA). The cells were suspended in ultra-low attachment surface 6-well culture plates (Corning Inc., Corning, NY, USA). The medium was changed every 3 days. The number of neurospheres was counted under a light microscope (Nikon Corp., Tokyo, Japan).

Spheroid formation assays were performed to verify the effect of miR-150 on cancer stem cells activation. SKpac-17 cells were transfected with pre-miR-150 and pre-miR-negative was added. Cells were plated at a density of 1000 cells/cm² in Ultra-Low Attachment Surface 6-well culture plates (Corning, Acton, MA, USA) in serum-free DMEM/F12 medium (Gibco/Life Technologies, Grand Island, NY, USA) supplemented with 20 ng/ml epidermal growth factor (Gibco, Carlsbad, CA, USA), 10 ng/ml basic fibroblast growth factor (Sigma–Aldrich, St. Louis, MO, USA), 0.4% bovine serum albumin (Sigma–Aldrich, St. Louis, MO, USA), and 5 μg/mL insulin (Sigma–Aldrich, St. Louis, MO, USA). Spheroid formation (50–100 cells per sphere) was assessed 7 days after seeding.