C1052

Cells were plated in 60 mm culture dishes at a density of 1×10⁶ cells per dish. After starvation for 12 h and treatment with the indicated conditions for 12 h, the treated cells were collected and washed twice with cold PBS. Then, a single cell suspension was fixed with 70 % cold ethanol for 12 h. Finally, according to the manufacturer’s instructions (C1052, Beyotime Biotechnology, China), the cells were stained with a propidium iodide (PI) mixture for 30 min at 37 °C before flow cytometry analysis (Beckman Coulter Flow Cytometer, Krefeld, Germany). The cell cycle distribution was further analyzed with ModFit LT (V4 1.7, Verity Software House, Topsham, ME).

Cells were plated in 60 mm culture dishes at a density of 1 × 10⁶ cells per dish. After being starved for 12 h and treated with the indicated conditions for 12 h, the treated cells were collected and washed twice with cold PBS. Then, the single cell suspension was fixed with 70% cold ethanol for 12 h. Finally, according to the manufacturer’s instructions (C1052, Beyotime Biotechnology, China), the cells were stained with a propidium iodide (PI) mixture for 30 minutes at 37 °C before flow cytometry analysis (Beckman Coulter Flow Cytometer, Krefeld, Germany). The cell cycle distribution was further analyzed with ModFit LT (V4 1.7, Verity Software House, Topsham, ME).

The cells were cultured with GSK-J4 for 24 hours in 6 cm dishes at a density of 4 x 10⁵ cells per dish. Cell cycle assay was performed according to the manufacturer’s instructions (Beyotime, C1052). Stained cells were applied to a BD FACSVerse flow cytometer (BD Bioscience), and data were analysed using ModFit software.

Cell cycle and apoptosis analysis were performed as described by the manufacturer (Beyotime Biotechnology, Shanghai, China, C1052). Cells were harvested via trypsinization, fixed in 75% ethanol, stained with propidium iodide solution at a final concentration of 50 ug/mL, and subjected to fluorescence-activated cell sorting (FACS) analysis.

After transfection for 48 h, the A549 and H1299 cells were harvested, washed twice with ice-cold PBS, and resuspended in 1× binding buffer. For cell apoptosis analysis, cells were incubated in Annexin-V/PI double staining solution (KGA101, KeyGen Biotech, Nanjing, China) for 20 min, according to the manufacturer's instructions, and then analysed using a FACSCalibur flow cytometer. The cell cycle was evaluated using a cell cycle and apoptosis analysis kit (C1052, Beyotime) according to the manufacturer's instructions using a FACS Calibur flow cytometer.

FRH-0201 cells were seeded at 2×10⁵ cells in a 6 cm cell culture dish and cultured overnight, after which the cells were exposed to one of three different treatments: varying concentrations of lapatinib(0, 1, 5, 20 μM) at 48 hours, constant concentration exposure (20 μM) for different time durations (0, 12, 24, 48 hours) and a combination of a varying concentrations of lapatinib(0, 1, 5, 10 μM) and a constant concentration of gemcitabine(0.5 μM) over a duration of 48 hours. The cell cycle distribution was detected by staining DNA with propidium iodide (C1052 Beyotime), while apoptosis and necrosis were detected using an Annexin V-FITC staining kit (C1062 Beyotime). The cell cycle distribution and apoptosis were determined through flow cytometry (Beckman CytoFLEX).