Annexin 5 fluorescein isothiocyanate 5 fitc propidium iodide

For the measurement of the cell apoptosis rate, Annexin (V‐fluorescein isothiocyanate) V‐FITC/propidium iodide (PI) (Beyotime) was utilized in lung cancer cells. In brief, transfected A549 and NCI‐H1299 cells were collected and washed with PBS (Invitrogen). Then, treated cells were incubated with 100 μl binding buffer containing 5 μl of an equal amount of Annexin V‐FITC/PI (Beyotime). FACSCalibur (BD Bioscience) was conducted to analyze the results of apoptosis. For assessment of cell cycle progression, transfected cells were treated with PBS (Invitrogen) twice, followed by fixation with ethanol. After additional incubation with RNase A (Sigma‐Aldrich) for 30 min, treated cells were incubated with PI for 30 min at room temperature. The percentages of cells in the G0‐G1, S, and G2‐M phases were detected according to the FACSCalibur flow cytometry (BD Biosciences). This assay was carried out at least three times.

In brief, transfected cells were harvested, and washed with PBS (Invitrogen), followed by fixation with ethanol for 1 h. After measurement with FACSan flow cytometry (BD Biosciences) with 5 μL Annexin (V-fluorescein isothiocyanate) V-FITC/Propidium Iodide (PI) (Beyotime, Nantong, China), apoptosis cells were analyzed with Quest software (BD Biosciences). For cell cycle assay, after treatment with ethanol and PBS (Invitrogen), cells were added to RNaseA and PI for 30 min at room temperature. Finally, FACSan flow cytometry (BD Biosciences) and ModFit LT 3.2 software (Verity Software House, Topsham, ME, USA) were utilized to analyze the percentages of cells in the G0-G1 and S phases.