*2.5. Apoptosis Assay*

LNCaP cells (6 × 10<sup>5</sup> cells) and PC-3 cells (4 × 10<sup>5</sup> cells) were seeded in 100 mm culture dishes (Sumitomo Bakelite Co., Ltd.). 24 h after seeding, the cells were treated with 0.1 or 0.3 mg/mL PFD, or vehicle (0.1% DMSO), for 48 h (LNCaP cells) or 72 h (PC-3 cells). After treatment, the cells were trypsinized, collected, and stained with annexin V–fluorescein isothiocyanate and propidium iodide simultaneously using the Annexin V-FITC Apoptosis Detection kit (BD Biosciences). The cell suspensions were analyzed using the BD FACS Canto II flow cytometer (BD Biosciences) to determine the percentage of apoptotic (annexin V–fluorescein isothiocyanate staining) and necrotic (propidium iodide staining) cells, as described previously [28]. A minimum of 20,000 cells were collected for all samples.
