Modfit version 4

For apoptosis, transfected A549 and H1975 cells were seeded into 6-well plates. Once they reached confluence (60-70%), the cells were collected and incubated with Annexin V-FITC (5 µl; Biogot Technology Co., Ltd.) and propidium iodide solution (5 µl; Biogot Technology Co., Ltd.) at room temperature for 15 min according to the manufacturer's instructions. Cells were subsequently suspended in 400 µl binding buffer (Biogot Technology Co., Ltd.). Early + late stage apoptosis was analyzed using a FACSAria flow cytometer (BD Biosciences). All data were analyzed with ModFit version 4.0 (Verity Software House, Inc.).

To evaluate cell apoptosis, SCC-9 or H357 cells (1×10⁵ cells) was seeded into 6 well plates. Once they reached confluence, cells were collected and incubated with Annexin V-FITC (5 µl) and propidium iodide (PI) solution (5 µl; Biogot Technology Co., Ltd.) at room temperature for 15 min according to the manufacturers' instructions. Cells were subsequently suspended in 400 µl binding buffer. To evaluate the cell cycle, SCC-9 and H357 cells were collected and fixed in 75% ethanol at −20°C overnight. Then, the fixed cells were washed with PBS and incubated with RNase A for 20 min at room temperature. These cells were stained with PI and incubated in the dark for 30 min at 4°C. Cell apoptosis and cell cycle progression were analyzed using flow cytometry (BD Biosciences). The percentages of cells within each phase of the cell cycle were analyzed with ModFit version 4.0 (Verity Software House, Inc.) and CellQuest version 5.1 (Thermo Fisher Scientific, Inc.).

For cell apoptosis, after transfection, MDA-MB-231 and MCF-7 cells were seeded into 6-well plates. Once they reached confluence (60-70%), cells were collected and incubated with Annexin V-FITC (5 µl) (Biogot Technology Co., Ltd.) and propidium iodide (PI) solution (5 µl; Biogot Technology Co., Ltd.) at room temperature for 15 min according to the manufacturer's instructions. Cells were subsequently suspended in 400 µl binding buffer (Biogot Technology Co., Ltd.). Cell apoptosis progression was analyzed using flow cytometry (FACSAria; BD Biosciences). All data were analyzed with ModFit version 4.0 (Verity Software House, Inc.)

The culture medium was discarded from a sample of 4×10⁵ HCCLM3 liver cancer cells. Cells were washed with phosphate-buffered saline (PBS) twice prior to digestion with a Trypsin/EDTA solution. Following this, cells were incubated at 37°C in an incubator containing 5% CO₂ until they were detached from the surface of the plate (~1 min). The cells collected were scattered with PBS and subsequently gathered by low-speed centrifugation (840 × g for 5 min at 4°C). Cells were mixed with 70% ice-cold alcohol and stored at −20°C overnight. The mixture was then centrifuged (840 × g for 5 min at 4°C) and the alcohol solution was removed to obtain the cell pellet. Following resuspension in PBS, 0.1% Triton-100, 4 mg/ml of RNase A (Beyotime Institute of Biotechnology) and 2 mg/ml of propidium iodide (PI) were added into the cells. Cells were then incubated at room temperature in the dark for 30 min, a 35-micron nylon mesh was included to filter the cell suspension. A flow cytometry instrument (Beckman Coulter, Inc., Brea, CA, USA) and cell cycle analysis software (Modfit Version 4.0; Verity Software House, Topsham, ME, USA) were used to analyze the cell cycle distribution in HCCLM3 liver cancer cells, according to the manufacturers' protocols.