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After 5 h of incubation, the Matrigel and unattached cells were removed, and the medium was replaced with fresh serum-free medium. 3.8. Western Blot Analysis {#sec3dot8-molecules-22-00702} -------------------------- The expression of uPAR was examined in isolated ASCs/PC3-NT and ASCs/PC3-Cyst. First, cells were cultured on a 12-well plate at 5 × 10^5^ cells/well for 3 days. Then, cells were lysed in M-PER buffer (Thermo Fisher Scientific, Waltham, MA, USA), containing protease and phosphatase inhibitors (Sigma-Aldrich). The protein concentration was determined with a DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were separated by SDS-PAGE, and were transferred to a PVDF membrane (Immobilon-P; Millipore, Billerica, MA, USA) in a buffer containing 25 mM Tris, 190 mM glycine, and 20% methanol. The PVDF membrane was blocked for 1 h with Tris-buffered saline (TBS) containing 5% nonfat dry milk. After washing with TBS containing 0.1% Tween 20 (TBST), the membrane was incubated with primary antibody (1:1000 in TBST) for 1 h at room temperature. After another washing step with TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The membrane was washed with TBST, and the immunoreactive bands were detected with ECL plus western blotting detection reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK). All western blot experiments were repeated three times independently. 3.9. 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