1–2 h incubation at 37uC, unattached cells were removed by rinsing the slides three times with PBS. The remaining attached cells were fixed and stained with Hema-3 (Fisher Scientific). Images in 5 different fields covering a majority of the area in each well of a 96-well plate were taken from all the treatment groups under a light microscope. The number of adhered cells from all the treatment groups was counted and the average was recorded for comparative quantification. Spheroid migration assay 4910 and 5310 spheroids were cultured in 96-well plates coated with 1% agar. Briefly, 36104 cells/well were seeded and cultured on a shaker at 100 rpm for 48 h in a humidified atmosphere containing 5% CO2 at 37uC. After the formation of spheroids, they were transfected with SV-sh, M-sh, U-sh, C-sh, MU-sh or MC-sh. 48 h after transfection, the spheroids were transferred to 24-well plates and incubated at 37uC. Twenty four hours after incubation, the spheroids were fixed and stained with Hema-3. Cell migration from the spheroids was assessed using light microscopy. The migration of cells from spheroids to monolayers was used as an index of cell migration and was measured using a microscope calibrated with a stage and ocular micrometer. Wound healing assay To study cell migration, we seeded xenograft cells (4910 and 5310) at a density of 26106 in a 6-well plate and transfected the cells with SV-sh, M-sh, U-sh, C-sh, MU-sh or MC-sh for 72 h. Then, a straight scratch was made in individual wells with a 200 mL pipette tip. This point was considered the ‘‘0 h,’’ and the width of the wound was photographed under the microscope. After 6–16 h, the cells were checked for wound healing and photographed at 16 h under the microscope. Wound healing was measured by calculating the reduction in the width of the wound after incubation. We also studied the effect of U-fl, M-fl and C-fl treatments in the absence and presence of a9b1 antibody on migration in 4910 cells by performing this assay. Matrigel invasion assay 4910 and 5310 cells were transfected with SV-sh, MU-sh or MC-sh for 72 h. Cells were trypsinized and 56104 cells were placed onto Matrigel-coated transwell inserts with 8-mm pore size. Cells were allowed to migrate through the Matrigel for 24 to 48 h. Then, cells in the upper chamber were removed with a cotton swab. The cells that adhered on the outer surface of the transwell insert and had invaded through the matrigel were fixed, stained with Hema-3, and counted under a light microscope as described previously [52]. Immunocytochemistry 4910 and 5310 cells (16104) were seeded on 2-well chamber slides, incubated for 24 h, and transfected with SV-sh, M-sh, U-sh, C-sh, MU-sh or MC-sh for 72 h. Then, cells were fixed with 10% buffered formalin phosphate and incubated with 1% bovine serum albumin in PBS at room temperature for 1 h for blocking. After the slides were washed with PBS, primary antibodies were added at a concentration of 1:100. The slides were incubated overnight at 4uC and washed thrice with PBS to remove excess primary antibody. Cells were then incubated with Alexa FluorH 594 (goat anti-mouse IgG, red) fluorescent-labeled secondary antibody for 1 h at room temperature. The slides were then washed thrice with PBS and covered with glass coverslips, and fluorescent photomi- crographs were obtained. Silencing Proteases in Glioma Immunohistochemistry Paraffin embedded brain sections (5 mm thick) from control and treatment groups were deparaffinized as per standard protocol. The sections were rinsed with PBS and treated with 1% BSA in PBS to prevent nonspecific staining and incubated with primary antibodies (1:50 dilution) at 4uC overnight. The sections were then washed in PBS and incubated with appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. After 1 h, sections were washed in PBS and incubated in DAB for 30 min. The slides were further washed with sterile water and subjected to dehydration. After dehydration, the slides were stained with hematoxylin to visualize the nucleus, mounted and observed under a light microscope. Immunohistochemical analysis for aVb3, a6b1 and a9b1 integrins was performed on the slide tissue microarrays (obtained from US Biomax, Inc., Rockville, MD, USA) of clinical GBM samples. FACS analysis 4910 cells (26106) were seeded on 100-mm tissue culture plates, incubated for 48 h, and then treated with 50 mM EDTA, washed with PBS, pelleted at 1000 rpm for 5 min, and resuspended at a concentration of 16106 cells/mL in PBS. Cells were then incubated with HRP-conjugated or fluorescence conjugated primary antibodies for 1 h on ice, pelleted, and washed three times with PBS to remove excess primary antibody. Cells that were incubated with HRP-conjugated primary antibodies were then resuspended in 1 ml of PBS and incubated with Alexa FluorH 594 (goat anti-mouse IgG, red) fluorescent labeled secondary antibody for 1 h on ice. After three more washes in PBS, cell pellet was resuspended in 10% buffered formalin and analyzed on a Coulter EPICS XL AB6064 flow cytometer (Beckman Coulter, Fullerton, CA). We also studied the effect of MU-sh and MC-sh treatments in 4910 cells on a9b1 integrin levels by performing FACS analysis. Clonogenic assay To study the effect of bicistronic plasmid shRNA treatments on proliferation, 4910 and 5310 cells were transfected with SV-sh, MU-sh or MC-sh. 72 h after transfection, cells were plated on 100-mm plates at 2000 cells per plate. Medium was subsequently changed every 3 days. After 2 weeks, the resulting colonies were fixed and stained with Hema-3, and counted as a measure of clonogenecity. Cell proliferation assay Cell growth was assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay. 4910 cells were transfect- ed with SV-sh, MU-sh or MC-sh. 72 h after transfection, 56103 cells were seeded in triplicate into 96-well plates and incubated different time intervals. Before each time point, we added 10 ml of 5 mg/ml freshly prepared MTT reagent to each well and continued the incubation for an additional 2 h to permit color development. After incubation, the crystals were dissolved in 2- propanol + hydrochloric acid solution and the plate was left in dark for 3 h. After 3 h, absorbance was measured using Biorad Microplate Reader. Intracranial administrations in nude mice The Institutional Animal Care and Use Committee of the University Of Illinois College Of Medicine at Peoria, Peoria, IL, USA approved all surgical interventions and post-operative animal care. 4910 and 5310 xenografts were trypsinized and resuspended in serum free medium at a concentration of 0.26105 cells/mL. Nude mice were injected intracerebrally with a 10 ml aliquot PLoS ONE | www.plosone.org 13 July 2010 | Volume 5 | Issue 7 | e11583