Cell proliferation assay

For the CCK-8 assay, A549 cells were seeded into 96-well plates at 1×10⁴ cells per well and cultured for 12 h using 10% FBS in DMEM. Cell proliferation was assessed under the guidance of the manufacturer's protocols of the Cell Counting Kit-8 (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). After transfection, we added 10 µl aliquot of CCK-8 solution to the test wells at 12, 24, 36, 48, and 60 h. Absorbance at 450 nm wavelength was measured after a 2 h incubation.

For the EdU assay, we seeded A549 cells into 48-well plates (Corning Incorporated, Corning, NY, USA). Until the transfected A549 cells reached 80% confluency, we used an EdU assay kit (Guangzhou RiboBio Co., Ltd.) to measure the cell proliferation rate. We followed the manufacturer's protocols except the nucleus staining dye was changed from Hoechst 33342 (supplied with the kit) to DAPI (Beyotime Institute of Biotechnology, Haimen, China) (23). When the stain was finished, the cells were imaged by fluorescence microscopy (BX51; Olympus Corporation, Tokyo, Japan).