In vitro cell behaviour assays

In all, 8X10⁵ cells were seeded in 6-well plates, grown to confluency, and scratched with a 200 μl pipette tip to make wounds of a consistent length before they were cultured in basal medium. After the cells were washed with PBS to remove dissociated cellular fragments, each wound was imaged at 0 and 24 h by inversion microscopy (Olympus, Japan). Cell migration was calculated using ImageJ.

In all, 3X10⁴ cells were seeded in the upper layer of the Transwell membrane, while the lower chamber contained 10% foetal bovine serum to induce cell migration. After the Transwells were incubated at 37 °C under an atmosphere with 5% CO2 for 24 h, the upper layer of the Transwell membrane was wiped, and the cells that passed through the membrane were stained with crystal violet for 20 min and observed by microscopy.

We seeded cells in 96-well plates at 2 × 10³ per well in 100 μl of complete medium and 10 μl of CCK-8 reagent (RiboBio, Guangzhou, China) for 1 h each day after 3 days of culture. We then used a microplate to measure the absorbance of each well at 450 nm. Each sample was evaluated in triplicate.

We used an EdU cell proliferation assay kit (RiboBio, Guangzhou; China). Cells were incubated with 200 μl of 5-ethynyl-20-deoxyuridine for 2 h at 37 °C and then fixed in 4% paraformaldehyde for 20 min. After that, the cells were permeabilized with 0.4% Triton X-100 for 10 min and incubated with Apollo® reagent (100 μl) for 30 min. Finally, the cells were stained with Hoechst for 30 min, and representative images were obtained using a Nikon inverted fluorescence microscope. The cell proliferation rate was calculated using the ratio of EdU-positive cells (red) to Hoechst-positive cells (blue).

In all, 2000 cells were seeded in 6-well plates and cultured in complete medium supplemented with 10% FBS. After 14 days, colonies were stained using crystal violet (Solarbio, Beijing, China) for 30 min and washed with PBS three times. Finally, we counted colonies with diameters greater than 1 mm.

GSCs were dispersed in Accutase solution and seeded in 6-well plates at 2000 cells per well. Microscopy was used to view images and measure the sphere diameters for quantification analysis.

GSCs were seeded in 96-well plates at densities of 0, 2, 4, 8, 16, 32 and 64 cells per well with 10 replicates per condition. After 14 days, the number of wells in which cells grew into colonies was calculated.