Low adherent 24 well culture plates

H460, H1299 and A549 cells were seeded in low-adherent 24-well culture plates (Corning, NY, USA) at 2 × 10³ cells per well, and incubated under serum-free condition in RPMI1640 (BioInd) containing 20 μl/ml of B27 (Invitrogen, CA, USA), 20 ng/ml of epidermal growth factor (EGF) (Invitrogen, Carlsbad, CA, USA), 20 ng/ml of basic fibroblast growth factor (bFGF) (Invitrogen, Carlsbad, CA, USA) and 1 % of penicillin-streptomycin (HyClone, Logan City, Utah, USA). After incubation at 37°C in a 5% CO2 incubator for 5-14 days, pictures were taken under a microscope and the number of spheres was counted in three separate 40× fields.

RCC cells were seeded in low-adherent 24-well culture plates (Corning, NY, United States) at 1 × 10³ cells per well, and incubated in DMEM/F12 (Gibco) containing 20 μl/ml B27 (Gibco), 20 ng/ml epidermal growth factor (EGF) (Gibco), 20 ng/ml of basic fibroblast growth factor (bFGF) (Gibco), 5 μg/ml insulin (Sigma), and 1% penicillin–streptomycin (Beyotime Biotechnology, China) under a serum-free condition. After 7 days of incubation at 37°C in a 5% CO₂ incubator, photographs were taken under a microscope and the number of cell spheres was counted in three separates in 100× fields. The cell sphere was defined when it reached a diameter ≥50 μm, and the differences in cell sphere number and size was determined mainly by comparing the number of cell spheres with diameters ≥50 μm or ≥100 μm between groups.