Hoechst staining kit

Cell apoptosis was detected using a TUNEL-FITC Apoptosis Detection kit and a Hoechst Staining kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), according to the manufacturer's protocol. The A549-F10, A549-mock and untransfected A549 cells, treated with paclitaxel, were seeded at a density of 1×10⁶ cells/ml onto coverslips, washed three times (5 min each) with PBS, fixed in 4% formaldehyde for 20 min, incubated in 70% ethanol at −20°C for 30 min, washed three times (5 min each) in PBS, and permeabilized in 0.1% Triton X-100/0.1% sodium citrate for 10 min at room temperature. Following another three 5-min washes with PBS, the cells were incubated with 3% H₂O₂ for 10 min at room temperature, followed by three washes in PBS for 5 min each. The cells were then incubated with a terminal deoxynucleotidyl transferase enzyme (BBI Life Science Corporation, Hong Kong, China) in the dark at 37°C for 90 min. Following two 2-min washes in PBS, the nuclei were stained with Hoechst 33258 in the dark at room temperature for 20 min. Finally, the cells were washed in the dark three times in PBS containing 0.5% Tween-20 for 2 min each, and were subsequently mounted in glycerol. The images were obtained using a fluorescence microscope (Nikon Corporation, Tokyo, Japan). The percentage of positive cells (as indicated by fluorescent staining) was calculated from the images.

After fixation with 4% paraformaldehyde for 20 min at room temperature, two CRC cell lines (HCT-116 and HT-29 cells) were used to observe nuclear changes and apoptotic body formation using the Hoechst staining kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol. Cells were visualized under a fluorescence microscope (IX53; Olympus Corporation; magnification, ×400) and counted by a professional researcher, who was blind to the grouping. The percentage of apoptotic cells was calculated as follows: Apoptotic cells (%)=(the number of apoptotic cells/the number of total cells) ×100%.