integrin, Rac1 were obtained from BD Biosciences (San Jose, CA); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from Novus Biologicals (Littleton, CO). Gelatin zymography MMP-9 activity in the conditioned medium was determined by gelatin zymography. 4910 and 5310 human glioma xenograft cells were transfected with SV-sh, M-sh, U-sh, C-sh, MU-sh and MC- sh for 72 h. Cells were washed and incubated with serum-free medium overnight. Equal amounts of protein were electropho- resed in 10% SDS-polyacrylamide gels containing 1.5 mg/mL gelatin. The gels were washed and gently shaken in three consecutive washings in 2.5% Triton X-100 solution to remove SDS. The gels were then incubated at 37uC overnight in incubation buffer [50 mmol/l Tris-HCl (pH 7.5), 0.05% NaN3, 5 mmol/l CaCl2 and 1 mmol/l ZnCl2]. Next, the gels were stained with 0.1% amido black in 10% acetic acid and 10% isopropanol and subsequently destained for 1 h. Gelatinolytic activities were identified as clear zones of lysis against a dark background. Fibrinogen zymography 4910 and 5310 human glioma xenograft cells were transfected with SV-sh, M-sh, U-sh, C-sh, MU-sh or MC-sh for 72 h. Cells were washed and incubated with serum-free medium overnight. Conditioned media was collected and zymography was performed to determine uPA activity. Equal amounts of protein samples were resolved on 10% SDS-polyacrylamide gels containing plasminogen and fibrinogen and stained with 0.1% amido black in 10% acetic acid and 10% isopropanol and subsequently destained for 1 h. Western blot analysis Xenograft cells were transfected with SV-sh, M-sh, U-sh, C-sh, MU-sh or MC-sh for 72 h. Cells were collected and lysed in RIPA buffer [50 mmol/ml Tris-HCl (pH 8.0), 150 mmol/ml NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS] containing 1 mM sodium orthovanadate, 0.5 mM PMSF, 10 m/ml aprotinin, 10 mg/ml leupeptin and resolved via SDS-PAGE. After overnight transfer onto nitrocellulose membranes, blots were blocked with 5% non-fat dry milk in PBS and 0.1% Tween-20. Blots were then incubated with primary antibody, followed by incubation with HRP-conjugated secondary antibody. Immunoreactive bands were visualized using chemiluminescence ECL Western blotting detection reagents on Hyperfilm-MP autoradiography film (Amersham, Piscataway, NJ). GAPDH (housekeeping gene) antibody was used to verify that similar amounts of protein were loaded in all lanes. Reverse transcription PCR analysis 4910 and 5310 cells were transfected with SV-sh, M-sh, U- sh, C-sh, MU-sh, MC-sh, U-fl, M-fl or C-fl. The cells were collected and total cell RNA was isolated. Approximately 500 ng of total RNA from each sample was synthesized into cDNA as per the manufacturer’s instructions using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Indianapolis, IN). We used the following sequences for the forward and reverse primers: N for MMP-9, 59CTCGAACTTTGACAGCGACA39 (forward) and 59GCCATTCACGTCGTCCTTAT39 (reverse); N for uPAR, 59GCCTTACCGAGGTTGTGTGT39 (forward) and 59CATCCAGGCACTGTTCTTCA39 (reverse); N for cathepsin B, 59GCTACAGCCCGACCTACAAA39 (for- ward) and 59CCAGTAGGGTGTGCCATTCT39 (reverse); Silencing Proteases in Glioma N for integrin aV, 59CACCAGCAGTCAGAGATGGA39 (for- ward) and 59GAACAACTGGCCCAACATCT39 (reverse); N for integrin a1, 59GTCCAGTTGGGAGAGGTGAA39 (for- ward) and 59ACTTGAAATGTGGGGCTGAC39 (reverse); N for integrin a2, 59CAAGTGGGATTCAGTGCAGA39 (for- ward) and 59GAGCACCAGCAACAAAGTGA39 (reverse); N for integrin a6, 59CCCCGCTGGTTATAATCCTT39 (for- ward) and 59GGTTTCCTCCATGCACACTT39 (reverse); N for integrin a7, 59GGGAACCAATACCCTGACCT39 (for- ward) and 59CTATAGCTGCTGGGGACTGC39 (reverse); N for integrin a9, 59CGGAATCATGTCTCCAACCT39 (for- ward) and 59CTCTGCACCACCAGATGAGA39 (reverse); N for integrin a10, 59CAACAGCATCTACCCCTGGT39 (for- ward) and 59GGTTCTTTGCTGCTCTCACC39 (reverse); N for integrin b1, 59CATCTGCGAGTGTGGTGTCT39 (for- ward) and 59GGGGTAATTTGTCCCGACTT39 (reverse); N for integrin b3, 59GCAATGGGACCTTTGAGTGT39 (for- ward) and 59GTGGCAGACACATTGACCAC39 (reverse); N for FAK, 59AGCTGATTGGAGTCATCACAGA39 (forward) and 59GAGGGTAGGAGGACAATTTGG39 (reverse); N for talin1, 59TGGAGAACTCAAAGGTGCTG39 (forward) and 59CGCCTTGATGGTCTTGACA39 (reverse); N for talin2, 59CACACTGGATTTTGGGGAGT39 (forward) and 59ATATCCTGGCCGAGAGGTG39 (reverse); N for profilin, 59AACGTTCGTCAACATCACG39 (forward) and 59TTTGGCAGCAATAAGGGGTA39 (reverse); N for vinculin, 59TACGAAGACAGGGGAAAGGA39 (forward) and 59TTGGAGCTGAGATGCAAGTG39 (reverse); N for actinin, 59GGCCAGTGATCTGTTGGAGT39 (forward) and 59TAGTCCTTCTGTCGCAGCAT39 (reverse); N for tensin, 59AGACACCCCTGTCTGCTCTG39 (forward) and 59GGGAAACTCCCCACTGAAG39 (reverse); N for Rac1, 59GGGAGACGGAGCTGTAGGTA39 (forward) and 59CAGCACCAATCTCCTTAGCC39 (reverse); N for Arp2, 59CTTCTGTTGCGAGGATACGC39 (forward) and 59AAGTTTTTCCACATCACCCTTC39 (reverse); N for GAPDH, 59AGCCACATCGCTCAGACACC39 (for- ward) and 59GTACTCAGCGGCCAGCATCG39 (reverse); N for b-actin, 59GGCATCCTCACCCTGAAGTA39 (forward) and 59GGGGTGTTGAAGGTCTCAAA39 (reverse); Reverse transcriptase PCR was set up using the PCR cycle [95uC for 5 min, (95uC for 30 sec, 55–60uC for 30 sec, and 72uC for 30 sec)630 cycles, 72uC for 10 min]. PCR products were resolved on a 1.6% agarose gel, visualized, and photographed under UV light. Adhesion Assay Adhesion was assessed as described previously [51] with modifications. 4910 and 5310 human glioma xenograft cells (16106) were seeded on 100-mm culture plates and incubated in a humidified atmosphere containing 5% CO2 at 37uC. 24 h after incubation, cells were transfected with SV-sh, M-sh, U-sh, C-sh, MU-sh or MC-sh. 72 h after transfection, cells were harvested by 50 mM EDTA treatment, washed with PBS, then resuspended in 10% serum-containing medium for 1 h at 37uC. Cells were washed twice with serum-free medium, resuspended in serum-free medium, and seeded at 30,000–50,000 cells/well in a 96-well plate coated with various components of extracellular matrix (ECM) such as collagen (type I), fibronectin, vitronectin or laminin. After PLoS ONE | www.plosone.org 12 July 2010 | Volume 5 | Issue 7 | e11583