Human epidermal growth factor (hegf)

Cells were suspended in serum-free DMEM/F12 (11320033, Gibco, NY, USA) supplemented with penicillin–streptomycin antibiotics, 20 ng/ml human epidermal growth factor (hEGF, 8916SF, Cell Signaling Technology, MA, USA), 20 ng/ml human fibroblast growth factor (hFGF, 33016015, Thermo Fisher, MA, USA), and 2% B27 Supplement (17504044, Gibco, NY, USA) without vitamin A. Subsequently, the indicated cells were cultured in ultralow attachment 24-well plates (3473, Corning, NY, USA) at a density of 1000 cells/well, and the size and number of cell spheres were evaluated via microscopy after 10 days.

Cells were seeded in six-well plates (5 × 10⁵ cells per well) and maintained for 12 h in a CO₂ incubator. To measure Rac-1, RhoA and CDC42 GTPases, they were treated with 10 µM NT4 in DMEM-0.1% BSA, serum-free, for 12 h at 37 °C and then incubated with 0.1 μg/mL hEGF (Cell Signaling) for 2 min for Rac-1 and CDC42 GTPases and for 5 min for RhoA GTPase. Cells were washed with cold PBS, lysed and processed for colorimetric G-LISA activation assays (Cytoskeleton, Denver, CO, USA), according to the manufacturer’s instructions. The activated forms of the G proteins were detected by incubation with specific primary antibody, followed by a secondary antibody conjugated with HRP and a detection reagent. The signal was read by measuring absorbance at 490 nm using a microplate reader. The experiment was repeated three times.