*2.6. ELISA*

For quantitative determination of TGFβ1 and PSA proteins, aliquots of conditioned medium from PCa cells were collected and subjected to ELISA using the Quantikine® human TGF-β1 immunoassay kit (R&D Systems, Inc., Minneapolis, MN, USA) and PSA Enzyme Immunoassay Test Kit (Hope Laboratories, Belmont, CA, USA), respectively.

### *2.7. Preparation of Cell Lysates*

LNCaP or PC-3 cells (1 × 106) were seeded in 100 mm culture dishes (Sumitomo Bakelite Co., Ltd.). 24 h after seeding, the cells were treated with PFD (0.1 or 0.3 mg/mL) or vehicle (0.1% DMSO) for 48 h. The cells were harvested by scraping, and whole cell lysates were prepared as described

previously [27]. Briefly, the cells were washed with ice-cold phosphate-buffered saline and lysed with CelLyticTM (Sigma-Aldrich Co.) containing 1% Nonidet P-40, 10 mM 4-(2-aminoethyl) benzensulfonyl fluoride, 0.8 mM aprotinin, 50 mM bestatin, 15 mM E-64, 20 mM leupeptin, and 10 mM pepstatin. After 60 min on ice, the lysates were centrifuged at 10,000 *g* for 10 min, and the supernatants were collected. The protein concentration was measured using the NanoDrop 2000 instrument (Thermo Fisher Scientific Inc.).
