Kga512

After cell transfection, cells were washed with PBS (centrifuged at 2000 rpm, 5 min) to collect and adjust the cell concentration to 1 × 10⁶/ml, and 1 ml of single cell suspension was taken for subsequent analysis. According to the manual instructions of the cell cycle detection kit (KeyGEN BioTECH, KGA512), we detected the cell cycle by flow cytometry. All data were analyzed by FlowJo software, V7.6.

After 72 hours of transfection of GNG4-shRNA, the cell cycle phase was evaluated by fluorescence activated cell sorting (FACS). First, the cells were treated with Triton X-100 and RNase, then the cell nuclei were stained with propidium iodide (PI), and finally, the DNA content was measured using a cell cycle kit (KGA512, KeyGen, China). At least 30,000 cells were analyzed in each experiment.

Cells were harvested, washed, and fixed in precooled 75% ethanol mixed with phosphate buffer saline (PBS) at 4°C overnight. The next day, the cells were washed and resuspended in 500 μl RNase and propidium Iodide (PI) mixed solution from a cell cycle detection kit (KeyGen Biotech, KGA512), and the cells were incubated at 37°C for 30 min for staining. All cell samples were harvested and analyzed on a flow cytometer.

Flow cytometry combined with a cell cycle kit (KGA512, KeyGEN BioTECH, Nanjing, China) were used to analyze the cycle distribution of U87 and U251 cells. Cells were seeded into 6-well plates, treated with different concentrations of GNE-477 (0, 0.5, 2 or 8 µM) for 48 h, digested with 0.25% trypsin and collected by centrifugation. Cells were washed with PBS once and fixed with 70% ice-cold ethanol, then moved to −20°C and incubated overnight. The fixative was removed by rinses with PBS before staining, and 500 µL of a preprepared PI/RNase A working solution were added and incubated in the dark for 30 min at room temperature. The final results were measured using a CytoFLEX flow cytometer (Beckman Coulter), and data were further analyzed using FlowJo 10.0.7 software.

The cells in each group were collected, digested with trypsin without EDTA, washed twice with PBS, and centrifuged at 1000 rpm for 5 min, to adjust the cell concentration to 1 × 10⁶ cells/ml. After discarding the supernatant, 500 µl of 70% chilled ethanol was added at 4 °C, and the cells were fixed overnight. On the following day, ethanol was removed by centrifugation, and the cells were washed three times with cold solution of PBS. Subsequently, 500 µl of the PI/RNase (KGA512, KeyGen Biotech, Nanjing, China) staining working solution that was prepared beforehand (PI:RNase A was prepared at 9:1) was added. Following staining at 4 °C for 30 min, flow cytometry was performed. The experiment was performed in triplicate.

Cells were washed by PBS once after digestion and centrifugation. For cell cycle, cells needed to be dissociated into single cells, fixed with 70% cold ethanol, and stained with PI (KeyGEN BioTECH, KGA512). For cell apoptosis, cells were incubated according to the steps of the cell apoptosis kit (Lianke Biotech, AP101). The stained cells were analyzed with the BD machine and FlowJo software.

Cells (1–2 × 10⁵) were seeded in 6-well plates and cultured for 24 h. Cells were digested and collected in a new EP tube and fixed them with cold ethanol at 4 °C overnight. After this, 500 µl propidium iodide (PI) and RNase A (1:9) were applied to incubate cells in the darkness (Cat. no. KGA512, KeyGen Biotech, Nanjing China). The results were analyzed by using a FACSCalibur flow cytometer (BD Bioscience). The percentage of different cell cycles was calculated using Graphpad Prism 6 software (GraphPad Software, Inc.).