Dna staining solution

Cellular ROS levels were measured using flow cytometry (Beckman, USA). Briefly, ACHN and Caki-1 cells were incubated with 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (Sigma‒Aldrich, USA) for 30 min at 37 °C. Next, the cells were washed three times with PBS and analyzed by flow cytometry. To analyze cell apoptosis, cells were stained with Annexin V FITC Apoptosis Assay Kit I (Sungene Biotech, China) according to the instructions, followed by flow cytometry. For the cell cycle assay, cells were stained with 1000 µl of 1 × DNA Staining Solution and 10 µl of permeabilization solution (Multi sciences, China).

The transfected PCa cells were sequentially digested and centrifuged. For cell cycle analysis, 1 × DNA staining solution (Multisciences, China) supplemented with propidium iodide was used to treat the cells for 30 min at room temperature. FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, USA) was used to stain cells for analysis of cell apoptosis. Then, the stained cells were incubated for 30 min at room temperature. The flow cytometry (cat. no. FC500; Beckman Coulter, USA) was applied in the analysis of cell cycle and cell apoptosis.

BLCA cells were analyzed for cell cycle distribution using flow cytometry (Beckman, USA) following incubation with 1 ml of 1× DNA Staining Solution (Multi Sciences, China) and 10 µl of permeabilization solution in the dark for 30 min.

Cell apoptosis and cell cycle were detected using Digital BD LSR II flow cytometry (BD Biosciences). For cell apoptosis analysis, cells at 40% of density were treated with different drug concentrations for 48 hours and then detected using Annexin V, FITC Apoptosis Detection Kit (Dojindo Molecular Technologies, Inc) following the manufacturer's protocol. For cell cycle progression assessment, 1 × 10⁵ cells were seeded into six‐well plates for 48 hours. Cells were then harvested and stored in 75% ethanol for 24 hours at −20°C and then mixed with DNA staining solution (Multi Science) in the dark for 30 minutes to detect cell cycle distribution.

To detect apoptotic cells, the pre-treated cells were harvested after trypsinization using EDTA-free trypsin, washed twice with PBS, and stained with the Annexin V-APC/7-AAD apoptosis kit (Multiscience, China) according to the manufacturer's instructions. The apoptotic cells were examined by flow cytometry (BD Bioscience, USA), and the data was analyzed with FlowJo X 10.0.7 software. For cell cycle analysis, the cells were harvested at about 80% confluency and fixed with cold 70% ethanol at -20°C overnight. After discarding the ethanol, the cells were washed with PBS, and incubated with DNA staining solution (Multiscience, China) for 30 minutes at room temperature. The DNA content were measured by flow cytometry, and the data was analyzed using ModFit software.

The cell cycle of NPSCs was detected by flow cytometry. Briefly, the samples were digested with 0.25% trypsin, collected, and then washed twice with PBS. Then, the cells were fixed in precooled 75% ethanol and stored at −20°C until further use. To analyze the cell cycle, the fixed cells were centrifuged and the ethanol was discarded. Then, the cells were hydrated with PBS for 15 min, and the supernatant was discarded after centrifugation. Then, 1 mL of the DNA staining solution (Multi Sciences, Hangzhou, China) was added and mixed well. The stained cells were incubated in the dark at room temperature for 30 min, followed by flow cytometry analysis.

The AGS and HGC-27 cells were seeded into 6-well culture plate with 2 × 10⁵ cells per well. After 48 h, the cells were collected, centrifuged and rinsed with precooling PBS twice. Then the cells were fixed with 75% ethanol at 4 °C overnight or -20 °C for long-term storage. Finally, the cells were centrifuged and added with 500 μl DNA staining solution (Multi Sciences, China) to stain the DNA. The stained cells were evaluated by flow cytometry (BD Calibur, San Jose, CA) and the data was analyzed by FlowJo 10.1.