2.8. TUNEL Assay

DNA fragmentation was detected via terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) assay using the fluorometric TUNEL system (G3250, Promega, Madison, WI, USA). HCT-15 and LoVo cells were seeded in 6-well plates at a density of 4 × 10⁵ per well and cultured for 24 h. After treatment with acetylshikonin (0, 1.25, 2.5, and 5 μM), cells were fixed with 4% formaldehyde solution for 25 min at 4°C and permeabilized using Triton X-100, diluted to 0.5% in PBS for 10 min. Apoptotic cells were treated with 25 μL TdT enzyme buffer. All cells were then stained using Hoechst stain solution (Sigma, St. Louis, MO, USA). Fluorescence-labeled damaged DNA strands were visualized using a fluorescence microscope (Nikon Eclipse TE 2000-U, Tokyo, Japan). Images were taken at 200x magnification [34].