Edu diluent

LUAD cells after transfection were seeded into the 96-well plate adding 100 μl of EdU diluent (Ribobio, Guangzhou China). Fixed cells were permeabilized in 0.5% Troxin X-100 and stained with 100 μl of 1 × Apollo^® 488, followed by DAPI staining. The proliferative cells were observed under the fluorescent microscope (Leica, Wetzlar, Germany).

Transfected cells were seeded into 96-well plates in 100 μl complete culture medium. The samples were incubated with 10 μl/well CCK-8 reagent (Dojindo, Kumamoto, Japan) for 2 h prior to each test. A microplate reader (Tecan, Männedorf, Switzerland) was employed to measure 450 nm absorbance every 24 h until 96 h.
Transfected cells were cultivated with 100 μl EdU diluent (Ribobio, Guangzhou, China) for 2 h. The cell samples were fixed in paraformaldehyde and sequentially stained with Apollo 567 working solution as well as Hoechst 33342 reaction solution in the dark. A fluorescence microscope (Leica, Wetzlar, Germany) was utilized to catch images.
Colony formation assay was employed to examine colony-forming ability of CCA cells. Transfected cells were equably seeded into 6-well plates and maintained in an incubator for 12 days. The cellular colonies were fixed in paraformaldehyde and stained with crystal violet (Beyotime). The colony numbers were reckoned via visual observation.