Propidium iodide staining solution

When the density of the primary myoblasts reached approximately 60%, they were transfected with miR-214 mimic, mimic NC, miR-214 inhibitor, and inhibitor NC, with three replicates in each group. After 24 h, the solution was changed. When the cell density reached approximately 90%, the cells were collected with trypsin and centrifuged to remove the supernatant. The cells were reconstituted with pre-cooled PBS, added to with pre-cooled absolute ethanol, and fixed overnight at −20 °C. After fixation, the supernatant was removed by centrifugation, and then the supernatant was centrifuged by pre-cooling PBS suspension cells. At the end of fixation, PBS and RNase a solution (Sangon Biotechnology, Shanghai, China) were added, evenly blown, and placed at 37 °C for 20 min. Afterward, a propidium iodide (PI) staining solution (Solarbio, Beijing, China) was added, followed by light avoidance at 4 °C for 30 min, and then tested on a machine. Flow cytometric analysis was performed on a FACSAria SORP flow cytometer (BD Company, Franklin, NJ, USA), and Modfit LT software 4.0 was used to process the data.

HCC cells were rinsed twice in prechilled PBS before being fixed overnight in 70% ethanol at 4 °C, followed by 50 µl of RNase A (Solarbio, China) to resuspend these cells. The cells were then stained with 200 µl of propidium iodide (PI) staining solution (Solarbio, China) at 37 °C for 60 min and 4 °C for 30 min in the dark. Cell cycle analysis was performed using a BD FACSCanto™ II flow cytometer (BD, USA). Furthermore, the apoptotic rate of cells was determined using a BD Pharmingen™ FITC Annexin V apoptosis detection kit® (BD, USA). A FACSCalibur flow cytometer (BD, USA) was used for apoptosis analysis after the cells were processed according to manufacturer instructions.