To determine cell apoptosis of A549-dereived stem cells and other cells, cells were collected at 80 g × 4 °C × 5 min for annexin-V/FITC (fluoresceine isothiocyanate)/PI (propidium iodide) staining after 48 h infection with the indicated plasmids or miRNAs. Briefly, cells were incubated with annexin-V and PI for 10 min and 5 min, respectively, in a dark room using the annexin V-FITC/PI staining kit (Beyotime, Beijing, China). Finally, the cell cycle was evaluated using the Beckman Coulter Navios EX flow cytometry (Beckman, Shanghai, China).
For analyzing cell cycle, the cells were cultured in 12-well plates (Thermo Fisher Scientific). After transfection with the indicated miRNAs or plasmids for 48 h, they were subjected to suspension and fixation in 75% ethanol at 4 °C for 12 h. Then, they were washed with PBS (phosphate‐buffered saline) for 3 times. Afterwards, they were resuspended in 500 μL staining buffer containing propidium iodide (5 mg/mL)/RNase (10 mg/mL) at 37 °C for 30 min in a dark room. Finally, cell apoptosis rate was evaluated using the flow cytometry. Each treatment group consisted four wells, and each assay was repeated for 3 times independently.
For analyzing cell cycle, the cells were cultured in 12-well plates (Thermo Fisher Scientific). After transfection with the indicated miRNAs or plasmids for 48 h, they were subjected to suspension and fixation in 75% ethanol at 4 °C for 12 h. Then, they were washed with PBS (phosphate‐buffered saline) for 3 times. Afterwards, they were resuspended in 500 μL staining buffer containing propidium iodide (5 mg/mL)/RNase (10 mg/mL) at 37 °C for 30 min in a dark room. Finally, cell apoptosis rate was evaluated using the flow cytometry. Each treatment group consisted four wells, and each assay was repeated for 3 times independently.