TUNEL and DAPI staining

U251 and U343 cells were seeded on a cover slide in a 24-well plate at a density of 3×10⁴ cells/well and maintained in a CO₂ incubator for overnight growth. Cells were treated with various concentrations of RA (0, 100, 200, and 400 µM) or vehicle for 24 h. Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining was performed according to the manufacturer's instructions (product no. C1086; Beyotime Institute of Biotechnology). Briefly, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min, incubated for 5 min with 0.3% Triton X-100 in PBS, and then incubated with a reaction mixture containing terminal deoxynucleotidyl transferase and fluorescent labeling solution for 60 min at 37°C according to the manufacturer's protocol. The cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at 37°C and mounted using ProLong™ Gold antifade reagent. Stained cells were analyzed using a confocal microscope (magnification, ×200).