Edu solution

Cell proliferation was determined using an Edu staining assay. Briefly, 50μmol/l EdU solution was prepared by diluting EdU solution (Solarbio Life Sciences, Beijing, China) with cell culture medium (1:1000). The treated endothelial cells were placed into 24-well plates and incubated with 100μl 50μmol/l EdU solution for 0.5 hour. After washing, the cells were fixed with methyl alcohol for 15 minutes and then decolored with 50μl 2mg/mL glycine for 5 minutes, after which the stained cells were examined.

For the CCK8 assay, 5000 transfected cells were planted and grown in 96-well plates. When the cells were cultivated for 24, 48 and 72 h, respectively, 10 μ CCK8 reagent (Biosharp, China) was added to the corresponding wells and reacted with the cells for 1 h. Finally, a microplate Reader (SpectraMax ABS, China) was utilized to analyze and record the absorbance at the 450 nm of these cells.
For the colony formation assay, 500 transfected cells were planted and cultured in 6-well plates. After 14 days, the colonies were recorded and counted after the cells were fixed and stained.
For the EdU assay, the transfected cells were immobilized for 15 min with paraformaldehyde after responding for 2 h to the EdU solution (Solarbio, China) of 50 μM at 37 °C, and the DAPI reagent (Sigma, USA) was employed to color the nucleus for 10 min. The representative images of EdU were then captured using a fluorescent microscope (Olympus BX53, Japan), and the EdU positive cells ratio was evaluated as the ratio of red versus blue fluorescent cells.

A549 and H1299 cells in each group were hatched in complete medium with 50 μmol L⁻¹ EdU solution (Solarbio, Cat. No. CA1170, Beijing, China) for 2 h. After fixing, cells were decolored using 50 μL glycine (2 mg mL⁻¹), and the cells were observed using a fluorescence microscope.