N., Brown V. MDA PCa2b were purchased from the American Type Culture Collection (Manassas, VA). LNCaP cells were routinely maintained in the regular medium, phenol red-positive RPMI 1640 medium supplemented with 5% FBS, 2 mm glutamine, and 50 g/ml gentamicin. The LNCaP cell model was described originally by Lin (30) and further characterized by Igawa (36). The C-33 cells are androgen-sensitive, whereas C-81 cells behave androgen independently. MDA PCa2b cells were maintained in BRFF-HPC1 medium supplemented with 20% FBS, 2 mm glutamine, and 50 g/ml gentamicin (37). Stable Transfectants with PAcP Expression Vector LNCaP C-81 cells were transfected with PAcP expression vector made up of WT PAcP cDNA with LipofectamineTM and PlusTM reagents in OptiMEM medium and followed the accompanying protocol. Two stable subclones designated as LNCaP-28 and -40 were described previously (30, 31). LNCaP-CMV is usually a subline of LNCaP C-81 cells transfected with the pCMV-neo vector alone. Generation of cPAcP siRNA and Establishment of cPAcP Knockdown Stable Subclones The pSUPER system-based siRNA approach and the protocol from OligoEngine (Seattle, WA) were utilized. Briefly, four different oligonucleotides were synthesized: siPAcP-78, 5-GCCTTAGCCTTGGCTTCTT-3; siPAcP-126, 5-GTGTACTAGCCAAGGAGTT-3; siPAcP-183, 5-GTCCCATTGACACCTTTCC-3; and siPAcP-236, 5-GGATTTGGCCAACTCACCC-3. A pair of 64-nucleotide oligonucleotides made up of 19 nucleotides targeting to PAcP mRNA sequence was inserted into the mammalian expression vector pSUPER at the BglII/HindIII restriction sites. Because siPAcP-126 consistently showed the high efficiency of PAcP knockdown, stable subclones were established in LNCaP C-33 cells by transfecting with siPAcP-126 pSUPER plasmid. Positive clones were selected by G418 (600 g/ml) and screened for cPAcP expression by Western blotting. Three subclones C-3, C-11, and C-17, were selected for further analyses. For the control, pSUPER vector made up of scramble oligonucleotide was transfected into C-33 cells, and clone V-3 was established. The transfection was performed as described above. Immunoblotting and Immunoprecipitation Subconfluent cells were harvested, and the immunoblotting was performed as described in previous reports (11, 30). For rehybridization, the membranes were stripped as described previously (11, 30, 31), blocked, and reprobed with specific Abs. For immunoprecipitation, cells were harvested and lysed in ice-cold cell lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Nonidet P-40, plus phosphatase and protease inhibitors). An aliquot of 250 g of lysate protein was incubated with primary Ab at 4 C overnight, and then protein A-agarose beads were added (20 l of 50% bead slurry) for 3 h at 4 C. The immunocomplexes were spun at 700 for 1 min, washed three times with ice-cold lysis buffer, and suspended in SDS-PAGE sample buffer. In Vitro Growth Kinetics Analysis Cells were seeded at densities as indicated in each set of experiments and cultured in the corresponding regular culture medium. The maintenance and determination of cell growth were conducted as described (30, 31). The cell number was counted with a Z1 model Coulter counter (Coulter Corp.). To determine androgen-independent growth, cells were maintained in a steroid-reduced medium for 4 days before cell number analysis (30, 33). Subfractionation of Cellular Proteins To subfractionate cellular proteins, subconfluent or confluent LNCaP C-33 cells were fed with a steroid-reduced medium for 2 days. Cell membrane, cytoplasmic, and nuclear proteins were fractionated following the protocol of the subcellular protein fractionation kit (Thermal Scientific). LNCaP C-33 cells were also subfractionated by ultracentrifugation. Briefly, confluent cells were lysed in a hypotonic Buffer A (20 mm acetate buffer, pH 5.0, containing 1 mm dithiothreitol and protease inhibitors), homogenized, and followed by centrifugation at 2000 for 1 h, and the supernatant was collected as the cytosol fraction. The pellets were suspended in Buffer B (Buffer.Biscardi J. Cell Culture Human PCa cell lines LNCaP and MDA PCa2b were purchased from the American Type Culture Collection (Manassas, VA). LNCaP cells were routinely maintained in the regular medium, phenol red-positive RPMI 1640 medium supplemented with 5% FBS, 2 mm glutamine, and 50 g/ml gentamicin. The LNCaP cell model was described originally by Lin (30) and further characterized by Igawa (36). The C-33 cells are androgen-sensitive, whereas C-81 cells behave androgen independently. MDA PCa2b cells were maintained in BRFF-HPC1 medium supplemented with 20% FBS, 2 mm glutamine, and 50 g/ml gentamicin (37). Stable Transfectants with PAcP Expression Vector LNCaP C-81 cells were transfected with PAcP expression vector made up of WT PAcP cDNA with LipofectamineTM and PlusTM reagents in OptiMEM medium and followed the accompanying protocol. Two stable subclones designated as LNCaP-28 and -40 were described previously (30, 31). LNCaP-CMV is usually a subline of LNCaP C-81 cells transfected with the pCMV-neo vector alone. Generation of cPAcP siRNA and Establishment of cPAcP Knockdown Stable Subclones The pSUPER system-based siRNA approach and the protocol from OligoEngine (Seattle, WA) were utilized. Briefly, four different oligonucleotides were synthesized: siPAcP-78, 5-GCCTTAGCCTTGGCTTCTT-3; siPAcP-126, 5-GTGTACTAGCCAAGGAGTT-3; siPAcP-183, 5-GTCCCATTGACACCTTTCC-3; and siPAcP-236, 5-GGATTTGGCCAACTCACCC-3. A pair of 64-nucleotide oligonucleotides made up of 19 nucleotides targeting to PAcP mRNA sequence was inserted into the mammalian expression vector pSUPER at the BglII/HindIII restriction sites. Because siPAcP-126 consistently showed the high efficiency of Lycopene PAcP knockdown, stable subclones were established in LNCaP C-33 cells by transfecting with siPAcP-126 pSUPER plasmid. Positive clones were selected by G418 (600 g/ml) and screened for cPAcP expression by Western blotting. Three subclones C-3, C-11, and C-17, were selected for further analyses. For the control, pSUPER vector made up of scramble oligonucleotide was transfected into C-33 cells, and clone V-3 was established. The transfection was performed as described above. Immunoblotting and Immunoprecipitation Subconfluent cells were harvested, and the immunoblotting was performed as described in previous reports (11, 30). For rehybridization, the membranes were stripped as described previously (11, 30, 31), ADRBK1 blocked, and reprobed with specific Abs. For immunoprecipitation, cells were harvested and lysed in ice-cold cell lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Nonidet P-40, plus phosphatase and protease inhibitors). An aliquot of Lycopene 250 g of lysate protein was incubated with primary Ab at 4 C overnight, and then protein A-agarose beads were added (20 l of 50% bead slurry) for 3 h at 4 C. The immunocomplexes were spun at 700 for 1 min, washed three times with ice-cold lysis buffer, and suspended in SDS-PAGE sample buffer. In Vitro Growth Kinetics Analysis Cells were seeded at densities as indicated in each set of experiments and cultured in the corresponding regular culture medium. The maintenance and determination of cell growth were conducted as described (30, 31). The Lycopene cell number was counted with a Z1 model Coulter counter (Coulter Corp.). To determine androgen-independent growth, cells were maintained in a steroid-reduced medium for 4 days before cell number analysis (30, 33). Subfractionation of Cellular Proteins To subfractionate cellular proteins, subconfluent or confluent LNCaP C-33 cells were fed with a steroid-reduced medium for 2 days. Cell membrane, cytoplasmic, and nuclear proteins were fractionated following the protocol of the subcellular protein fractionation kit (Thermal Scientific). LNCaP C-33 cells were also subfractionated by ultracentrifugation. Briefly, confluent cells were lysed in a hypotonic Buffer A (20 mm acetate buffer, pH 5.0, containing 1 mm dithiothreitol and protease inhibitors), homogenized, and followed by centrifugation at 2000 for 1 h, and the supernatant was collected as the cytosol fraction. The pellets were suspended in Buffer B (Buffer A plus 0.15 m NaCl) and spun at 100,000 for 45 min. The supernatant was designated as the membrane-associated fraction. The pellet was then solubilized in Buffer C (Buffer B.
