Propidium staining solution

After culturing, the cells were digested with trypsin and fixed in 1 mL of 70% ethanol at 4 °C for 2 h. Following centrifugation at 1000× g for 3 min, the supernatant was removed, and 1 mL of pre-chilled PBS was added. Propidium staining solution (C1052, Beyotime) was prepared, and 0.5 mL of the staining solution was added to each sample, followed by incubation at 37 °C in the dark for 30 min. The samples were placed on ice and then flow cytometry analysis was performed. This was repeated three times for each set (n = 3).

ICP2 cells were seeded in 6-well plates and cultured in basal medium. At 48 h post-transfection, cells were harvested by centrifugation at 1,000 × g for 5 min. After washing with cold phosphate buffered saline (PBS) (Gibco), the cells were fixed in cold 70% ethanol at 4 ℃ overnight. Subsequently, the cells were re-collected by centrifugation at 1,000 × g for 5 min and incubated with propidium staining solution (Beyotime) at 37 ºC for 30 min in the dark conditions, according to manufacturer’s instructions. The cell cycle was analyzed by flow cytometry using a BD FACSCalibur flow cytometer (BD Biosciences, San Diego, CA, USA), and the data were analyzed using the FlowJo_v10.8.1 software.