Modfit version 3.1

In accordance with the cell cycle kit instructions (Beyotime, Shanghai, China), embryonic myoblasts were seeded into the 6-well plates cultured in GM, and were transfected for 24–48 h. Briefly, the cells were digested by trypsin to be collected into a 1.5 mL centrifuge tube, then pre-cooled 70% ethanol was added to cells for fixation overnight at 4 °C. The propidium (PI) staining solution was prepared according to the instructions to dye the cell samples in darkness at 37 °C for 30 min. The DNA content of the cells was detected by the BD LSRFortessa flow cytometer (BD, Franklin Lakes, NJ, USA) after PI staining, and the distribution of DNA content was analyzed using Modfit (Version: 3.1) (Topsham, ME, USA).

Flow cytometry was performed using a Cell Cycle kit (Beyotime, Shanghai, China). In short, trypsin was employed for cell digestion. After embryonic myoblasts were transfected for 24–48 h, the cells were gathered in a centrifuge tube with a capacity of 1.5 mL. Subsequently, 70% ethanol, which had been pre-cooled at 4 °C, was added into the centrifuge tube to preserve the cells for a duration of 12 h. Afterward, propidium (PI) staining was employed to color the cell samples in a dark environment at a temperature of 37 °C for 30 min. In the end, the DNA content of the cells was detected using Modfit (Version 3.1) (Topsham, ME, USA) on a BD LSRFortessa flow cytometer (BD, Franklin Lakes, NJ, USA).