Gels were washed in 2

Gels were washed in 2.5% Triton X-100 to remove SDS and followed by overnight incubation at 37 C in Tris-CaCl2 buffer (pH 7.6). suppressed tumor sections was associated with decreased co-localization of integrin-V3 and MMP-2. In summary, these data provide new insights into the mechanisms underlying MMP-2-mediated VEGF expression in lung tumor angiogenesis. and models. Our findings indicate that MMP-2 transcriptional inactivation significantly reduced integrin-V3-mediated, PI3K/AKT-induced VEGF expression, which ultimately decreased tumor cell-induced angiogenesis. MATERIALS AND METHODS Cells and Reagents A549 and H1299 lung adenocarcinoma cells were cultured in RPMI 1640 (ATCC Manassas, VA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 50 units/mL penicillin, and 50 g/mL streptomycin (Life Technologies, Inc., Frederick, MD). For human microvascular dermal endothelial cells (HMEC-1), glutamine, EGF and hydrocortisone (Stem Cell Technologies, British Columbia, Canada) were added to Advanced MEM medium (Invitrogen, Carlsbad, CA). Cells were incubated at 37 C in a humidified 5 % CO2 atmosphere. We used antibodies specific for MMP-2, VEGF (VEGF-A), VEGFR-2, HIF-1, GAPDH, integrin-V3 (Clone 23C6), MT1MMP, Q203 (Santa Cruz Biotechnology, Santa Cruz, CA), PI3K, AKT, phospho-AKT (Ser-473) (Cell Signal Technology, Boston, MA), functional blocking integrin-V3 (Clone 23C6; Cat# CBL544; Millipore Corporation, Temecula, CA) and anti-Human Von-Willebrand Factor (Factor-VIII; Dako North America, Inc, CA), and HRP/Alexa Fluor? conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). We also used constitutively active-AKT (myr-AKT) plasmid (Addgene, Plasmid 10841), human recombinant-MMP-2, human recombinant-VEGF165 (Millipore Corporation, Temecula, CA), human VEGF quantikine ELISA Kit, human MMP-2 quantikine ELISA Kit (R&D Systems, Minneapolis, MN) and ARP-100, a MMP-2 specific inhibitor (TOCRIS Bioscience, Ellisville, MO) in this study. Adenoviral siRNA constructs and infection Adenoviral siRNA constructs for MMP-2 (Ad-MMP-2-Si) and scrambled vector (Ad-SV) were constructed and amplified as described by us previously.27 Viral titers were quantified as pfu/mL following infection of 293 cells. Titers were obtained for Pecam1 Ad-SV (7.6 1011 pfu/mL) and for Ad-MMP-2-Si (5.0 1011 pfu/mL). The amount of infective adenoviral vector per cell Q203 (pfu/cell) in culture media was expressed as multiplicity of infection (MOI). Virus constructs were diluted in serum-free culture media to the desired concentration, added to cells, and incubated at 37 C for 1 h. The necessary amount of complete medium was then added and cells were incubated for the desired time periods. Gelatin zymography Tumor conditioned medium was prepared as follows: after 36 h of mock, 100 Q203 MOI of either Ad-SV or Ad-MMP-2-Si infection, medium was removed from A549 cells, washed with PBS, 3 mL of serum free medium was added, and cells were incubated for another 12 h. MMP-2 secretion into conditioned medium was determined by gelatin zymography as described previously.27 Briefly, conditioned medium containing equal amounts of protein was resolved over gelatin-SDS-polyacrylamide gels. For immunoprecipitated samples, immunocomplexes were incubated with 1X sample buffer for 30 min at RT and were resolved over gelatin-SDS-polyacrylamide gels. Gels were washed in 2.5% Triton X-100 to remove SDS and followed by overnight incubation at 37 C in Tris-CaCl2 buffer (pH 7.6). Gels were stained with Coomassie brilliant blue and subsequently de-stained for 1h. Gelatinolytic activities were identified as clear zones of lyses against a dark blue background. Immunoprecipitation and western blotting A549 cells were infected with 100 MOI of either Ad-SV or Ad-MMP-2-Si for 48 h. Whole cell lysates were prepared by lysing cells in radioimmunoprecipitation assay (RIPA) lysis buffer with proteinase inhibitors. For Q203 the hypoxic experiments, cells were infected for 36 h followed by incubation at 1 % O2 for 12 h in serum-free medium. Equal amounts of protein fractions or immunoprecipitates of lysates with indicated antibodies.