Matrigel reconstituted basement membranes

Human prostate cancer cells PC-3, LNCaP, DU145 and monocytoid U-937 and THP-1 cell lines were purchased from ATCC, Rockville, Maryland. The PC-3 High and Low Invasive cell lines were selected by three serial passages through Matrigel reconstituted basement membranes (Becton Dickinson, Lincoln Park, NJ) in a Transwell chamber with 8 μM pore size [31 (link)]. The selected cells were placed in co-cultures with monocyte-lineage U-937 or THP-1 cells at standard seeding densities. For transfection experiments, the prostate cancer cells were exposed to 5 μg of dominant negative pEGFP-IκBα S32/S36 expression vector or control vector pEGFP-C1 (Clontech, Mountain View, CA) [32 (link)]. All cells were maintained in a humidified atmosphere of 5% CO₂ at 37°C in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Biofluids, Rockville, MD); 2 mM Lglutamine; 100 units/mL penicillin and 100 μg/mL streptomycin (Life Technologies, Inc.). Bay11-7082 and RS102895 HCl were obtained from Sigma-Aldrich, St. Louis, MO. Lactacystin was purchased from Biomol (Enzo Life Sciences, Inc.), Plymouth Meeting, PA. Gefitinib (N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine) was purchased from Tocris Bioscience. Recombinant human CCL2 (279-MC), anti-human CCL2 neutralizing antibodies (MAB279) and IL-8 (208-IL) were purchased from R&D Systems, Inc., Minneapolis, MN.