Accuri c6 software v1

For the cell cycle analysis, 2×10⁵ A549-shLuc and A549-shCNTN1-1 cells were seeded in a 6-well plate. The cells were subsequently harvested and washed with PBS, then fixed with 70% ethanol for 30 min at room temperature. The fixed cells were then stained with fresh propidium iodide (PI) containing RNase A for 30 min at 37°C. Finally, a total of 1×10⁴ cells were analyzed from each sample using a BD Accuri C6 flow cytometer (BD Biosciences).
For apoptosis analysis, the transfected cells were seeded in a 6-well plate (2×10⁵ cells/well) and treated with 12.5 µM gefitinib. After incubation for 48 h, the cells were digested with trypsin, washed with cold PBS twice, resuspended in 500 µl binding buffer, then incubated with 5 µl FITC-Annexin V and 5 µl PI for 15 min at room temperature, in the dark. Flow cytometry was performed using a BD Accuri C6 flow cytometer (BD Biosciences) following the manufacturer's instructions, to detect cell apoptosis by determining the relative amount of Annexin V-FITC-positive, PI-negative cells or FITC-Annexin V-positive and PI-positive cells. The cell cycle and apoptosis data analysis was performed using BD Accuri C6 Software v.1.0.264.21 (BD Biosciences).