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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

United statesEteläuomen läiCell signaling technologyEthics committeeChinese academy of sciences shanghaiChongqing medicalFirst affiliated hospitalChongqing medical universityChinese academyChina keygenMassachusetts united statesWhitneyu testImmunohistochemical expressionClinicopathological featuresInvasive behaviorGene ontology