2.8. Hoechst 33342/PI staining assay

BCPAP cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and cultured overnight. Then the cells were pretreated with or without 4-PBA (10 mM) for 1 hour followed by curcumin (50 μM) treatment for additional 24 hours. After incubation, the cells were stained with a combination of Hoechst 33342 (10 μg/mL) and PI (10 μg/mL) at 37 °C for 15 minutes. The cell images were captured using a fluorescence microscope (Olympus, X51, Japan). Cell were counted for five independent microscopic fields per well. Cell death rate (%) = (PI-positive staining cell number/total cell number) × 100%.