Cell cycle and apoptosis assay kit

After transfection, T24 and EJ cells were digested with trypsin, rinsed three times with cold PBS and resuspended as single cells, and the cells were treated accordingly according to the instruction manual for the Cell Cycle and Apoptosis Assay Kit (C1052; Beyotime Institute of Biotechnology, Shanghai, China). processing. Flow cytometry (BriCyte E6 system) was used to analyze the cell cycle and apoptosis processes, and the results were statistically analyzed using FlowJo 10 software.

The Cell Cycle and Apoptosis Assay Kit (Beyotime, Shanghai, China) was used to assess the effect ofAZD5153; AZD5153, BMN673, and the combination of the two drugs on the cell cycle of HCT116 and LoVo cells, following the manufacturer's instructions. The cells were seeded into 6-well plates at a density of 2 × 105 cells/well. First, we treated HCT116 with 2μmol/L and LoVo1μmol/L with cells with single drug AZD5153.Then we treated HCT116 with 2μmol/L AZD5153 and 0.25μmol/L BMN673 (individually and in combination), while LoVo cells were treated with 1μmol/L AZD5153 and 0.5μmol/L BMN673 (individually and in combination). The cell cycle was measured by flow cytometry 48 h after treatment.

At the end of the BHBA treatment, GCs were collected with 0.25 % trypsin and then washed with pre-chilled PBS. Next, 1 ml of pre-chilled 70 % ethanol was added to fix the cells for 2 h at 4°C. The cells were then collected by centrifugation at 1,000 g for 5 min. A Cell Cycle and Apoptosis Assay Kit (Beyotime, C1052) was used for this experiment. Propidium iodide (PI) staining solution was prepared according to the manufacturer's instructions; then 0.5 ml of PI was added to each tube of cell samples. The GCs were then incubated for 30 mins in the dark at 37 °C. Finally, red fluorescence was detected by flow cytometry at an excitation wavelength of 488 nm. The results were analyzed by FlowJo software (version, 10.4).

The cultured myobalsts in growth media were collected 48 h after transfection. The cells were then fixed overnight at 4 °C in 70% ethanol, following the protocol of the Cell Cycle and Apoptosis Assay Kit (Beyotime, Shanghai, China). Subsequently, the cell precipitate was treated with propidium iodide (PI) solution and incubated at 37 °C for 30 min in the dark. Finally, the samples were analyzed using Beckman Coulter flow cytometry (Indianapolis, IN, USA).