Ultra low cluster plate

The U251 and U87-MG cells in good growth condition were counted and 1 × 10³ cells were plated into 12-well Ultra Low Cluster plates (Corning). The cells were cultured with a serum-free DMEM/F12 medium containing 100 U/ml penicillin, 100 μg/ml streptomycin, 20 ng/ml epidermal growth factor (EGF), 10 ng/ml basic fibroblast growth factor (bFGF) (Gibco, USA) at 37 °C for 10–14 days. Three replicates were set for each type of cell. The culture medium was replaced according to the cell growth rate and the color changes of the culture medium. The cultures were photographed regularly under the microscope to observe tumor-sphere formation.

Cells were seeded in ultra-low cluster plates (Corning Inc., Corning, NY) and cultured in DMEM/F12 serum-free medium (Invitrogen) supplemented with 20 ng/mL EGF, 20 ng/mL bFGF, 0.4% BSA, and 2% B27 (BD Pharmingen, Carlsbad, CA) as well as 1% methyl cellulose (Sigma-Aldrich). For ELDA, the cells at different densities were cultured 12 wells per cell density in stem cell medium in 96-well plates for 1 to 2 weeks. The numbers of wells with at least one tumorsphere (diameter >50 μm) were counted in a blinded manner. The frequency of sphere-initiating cells was calculated by ELDA online program at http://bioinf.wehi.edu.au/software/elda/.

The generation of otic spheres followed previously described protocols (Oshima et al., 2009 (link)) with modifications during differentiation phase to test GSIs. Briefly, OCs were dissociated and single cells were deposited in ultralow-cluster plates (Corning) and expanded in “full otic medium” consisting of DMEM/F12, supplemented with N2, B27 (Thermo Fisher), EGF (20 ng/ml; Chemicon), bFGF (10 ng/ml; Chemicon), IGF-1 (50 ng/ml; Chemicon), and heparan sulfate (50 ng/ml; Sigma-Aldrich). After culturing for 3–4 days at 37°C, spheres were collected, plated in cell culture-treated 96 well-plate and cultured in DMEM/F12, supplemented with N2, B27 with/without GSIs for 7 days after which they were lysed for RNA extraction.

For sphere formation culture, the cells were incubated in Dulbecco’s modified eagle’s/F12 serum-free medium (Welgene Inc.) supplemented with 2% B-27 (Gibco; Thermo Fisher Scientific Inc.), 20 ng/mL recombinant human epidermal growth factor (Gibco; Thermo Fisher Scientific Inc.), and 20 ng/mL recombinant human fibroblast growth factor basic (Gibco; Thermo Fisher Scientific Inc.) in six-well ultra-low cluster plates (Corning Inc.). Once the diameters of the formed spheres reached 50 µm, the spheroids were transfected with miR-31-3p or control miRNA at a final concentration of 80 nM using G-fectin (Genolution Pharmaceuticals Inc.) according to the manufacturer’s protocol. Four days after transfection, images of the spheres were captured using an Olympus CKX52 microscope (magnification, ×40), and the number and diameter of the spheres were measured in four randomly selected fields. Experiments were performed in triplicate and repeated three times.

Cells (500 cells/well) were seeded into 6-well Ultra Low Cluster plates (Corning) and cultured in suspension in serum-free DMEM-F12 (BioWhittaker), supplemented with B27 (1:50, Invitrogen), 20 ng/mL endothelial growth factor (EGF; BD Biosciences), 0.4% bovine serum albumin (Sigma), and 4 mg/mL insulin (Sigma). After 10–12 days, the number of cell spheroids (tight, spherical, non-adherent masses > 50 μm in diameter) were counted, and images of the spheroids were scored under an inverse microscope (spheroids formation efficiency=colonies/input cells × 100 %).

Cells (500 cells/well) were seeded into 6-well Ultra Low Cluster plates (Corning) and cultured in suspension in serum-free DMEM-F12 (BioWhittaker), supplemented with B27 (1:50, Invitrogen), 20 ng/ml endothelial growth factor (EGF; BD Biosciences), 0.4% bovine serum albumin (Sigma), and 4 mg/ml insulin (Sigma). After 10–12 days, the number of cell spheroids (tight, spherical, non-adherent masses > 50 μm in diameter) were counted, and images of the spheroids were scored under an inverse microscope (spheroids formation efficiency = colonies/input cells× 100%).