Cell Proliferation Assay
The cell proliferation reagent CCK-8 (Monmouth Junction, NJ, USA) was used to assess the proliferation of FaDu cells. In short, cells transfected as described above and incubated for 48 h were added to 96 well plates (1×10
3 cells/well) containing 10% FBS medium. Ten microliters (10 μL) of CCK-8 were added to cells and cells were incubated at 37°C for 1 h. Finally, the optical density at 450 nm was measured using a microplate reader (iMark Microplate Absorbance Reader; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Colony Formation Analysis
The colony-forming assay was used to detect cell proliferation. Forty-eight hours (48 h) after transfection, 1000 cells per space were inoculated into six empty plates and incubated in medium containing 10% FBS for about 2 weeks. The medium was changed every three days. Finally, cells were fixed with 100% formaldehyde and stained with 0.5% crystal violet. The number of colonies was counted under an inverted microsc