LSM510 software using a scan zoom of 1.7 and compiled in Adobe Photoshop. Quantitative RT-PCR analysis Cells infected with non-target or shEcad-expressing viruses were rinsed with 1xPBS. A portion of the cells on each plate were scraped up using a cell lifter and transferred in PBS to a sterile microfuge tube. The remaining cells were returned to culture conditions for future sampling. For harvests in which both protein and RNA were to be isolated, the cell sample was divided in half prior to collecting the cells by centrifugation; the individual cell pellets were then lysed for either RNA or protein isolation. Total RNA was isolated using the miRCURY RNA Isolation Kit - Cell and Plant (Exiqon Inc., Woburn, MA) according to manufacturer protocol. Equal ng amounts of total RNA were converted to cDNA using the High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems Inc., Foster City, CA). qPCR reactions were performed in triplicate with 10 ng of cDNA and TaqManH FAST Universal PCR master mix (Applied Biosystems). Human CDH1 (ABI# Hs00170423_m1) and human GAPDH (ABI# Hs99999905_m1) primer/probe sets were purchased from Ap- plied Biosystems. All amplification data were collected with an Applied Biosystems Prism 7900 sequence detector and analyzed with Sequence Detection System software (Applied Biosystems). Data were normalized to GAPDH, and mRNA abundance was calculated using the DDCT method [60]. Samples with an average GAPDH CT value greater than 1 CT (1 fold-change) away from the wild-type GAPDH CT value were excluded from further analysis. Boyden chamber migration assay Migration assays were performed on 10 mm polycarbonate membrane, 8 mm pore size, transwell culture inserts (upper chamber) (Nalge Nunc International, Rochester, NY) coated with 15 mg/ml Bovine Type I collagen (BD Biosciences) and placed in 24 well plates (lower chamber). The day before the assay, culture medium on the cells was changed to DMEM containing 250 mg/ ml BSA, with additional 2 mM L-glutamine and 1% non-essential amino acids, and the cells incubated overnight at 37uC, 5% CO2. The day of the assay, cells were harvested by a very brief incubation in 0.25% Trypsin-EDTA, counted using Trypan Blue exclusion to determine the number of live cells, and resuspended in DMEM/BSA at 56105 cells/ml. 16105 cells were placed in the upper chamber; DMEM/BSA containing 0.5% Fetal Bovine Serum was placed in the lower chamber. Cells were allowed to migrate to the underside of the transwell for 6 hours at 37uC, 5% CO2. Any cells that remained in the upper chamber were removed by gently scrubbing with cotton swabs. Cells on the underside of the transwell chambers were collected by incubating the bottom of the chamber in 225 ml of Cell Dissociation Buffer (equal parts 0.5% phenol red-free Trypsin EDTA (Invitrogen) and Hank’s Balanced Salt Solution (5.37 mM KCl, 0.45 mM KH2PO4, 137 mM NaCl, 4.17 mM NaHCO3, 0.34 mM Na2HPO4, 5.55 mM D-glucose)) for 20 minutes at 37uC, 5% CO2. At the end of this time 75 ml of a 4x Lysis+Dye Solution (20x Lysis Solution and 400x GR DNA Binding Dye, both from the CyQuant Cell Proliferation Assay kit (Invitrogen), diluted in water to make a 4x stock solution) was added to the cell-containing samples in the lower chamber under each transwell. Samples were frozen at 280uC for at least 1 hour. They were then transferred to a black, clear-bottom, 96-well plate and sample fluorescence read by a SpectraMax M5 plate reader running SOFTmax PRO software (Molecular Devices Corporation, Sunnyvale, CA) with excitation at 480 nm and absorption at 520 nm. Sample fluorescence for each cell type was normalized to the fluorescence E-Cadherin Expression in GBM obtained from 0.256105 cells plated directly in triplicate wells of a 24 well plate, allowed to adhere for 6 hours at 37uC, 5% CO2, and then quantified by the same method (incubation with Cell Dissociation Buffer and 4x Lysis+Dye Solution followed by one round of freeze-thaw lysis). This quantification method measures DNA concentration to determine the relative number of cells in each sample. Data were expressed as percentage of control (non- target shRNA-transduced) cell migration and are presented as the mean +/2 SEM of 4 independent experiments performed in triplicate. Cell growth assay MTT cell growth assays were carried out by plating 4000 cells/ well in 96 well plates, with triplicate wells for each harvest. Cells were allowed to grow for 24, 48, 72, 96, or 120 hours. Prior to each harvest, cells were incubated with 1 mg/ml MTT (Sigma- Aldrich) at 37uC, 5% CO2 for 1 hour. Formazan precipitate (produced in proportion to the number of live cells) was then extracted from the cells by replacing the media on the cells with 200 m/well DMSO (Sigma-Aldrich), pipetting up/down, and incubating at 37uC, 5% CO2 for 5 minutes. The absorbance at 550 nm was then determined for each sample. Data were plotted as mean +/2 SD. This experiment was performed three times in triplicate; a representative plot is presented. Statistics Overall and recurrence-free survivals were evaluated using the Kaplan-Meier method, performed with SAS software (SAS Institute Inc., Cary, NC). Unpaired, one-tail t test (Figure 3), two-way ANOVA and Bonferroni post hoc testing (Figure 6C), and the paired t test (Figure 6D) were done using GraphPad Prism 4 software (GraphPad Software Inc., La Jolla, CA). Supporting Information Figure S1 Comparison of anti-human E-cadherin antibodies for use with immunohistochemistry. Immunohistochemistry for E- cadherin expression was carried out using either the 4A2C7 (Zymed/Invitrogen) or clone 36 (BD Transduction Labs) antibod- ies on the Mayo Clinic GBM-A3 TMA. Among other samples, this TMA includes non-neoplastic brain tissue (both gray and white matter), a sample of infiltrating ductal carcinoma (IDC) breast tumor, and a plug of U251 glioma cells. The scale bar is 20 mm and applies to all images. Found at: doi:10.1371/journal.pone.0013665.s001 (3.49 MB TIF) Figure S2 Examples of minimally versus highly invasive orthotopic GBM xenografts. H&E stain of mouse brain orthotop- ically implanted with GBM xenograft line 12 (minimally invasive) or GBM line 8 (highly invasive). Left images (12.5x magnification) show a section of the entire brain; right images (200x) are magnified to demonstrate the tumor/normal brain interface of the two GBM lines. Left image scale bars are 1 mm; right image scale bars are 50 mm. Found at: doi:10.1371/journal.pone.0013665.s002 (4.05 MB TIF) Figure S3 Comparison of E-cadherin shRNA effects on SF767 and MCF7 cells. A. SF767 cells were infected with freshly prepared, high titer virus expressing non-target shRNA or shEcad#21, #22, or #23. CDH1 mRNA levels in these cells were quantified using qPCR at days 2, 4, and 6 post infection (p.i.; top); E-cadherin protein expression was determined at days 4 and 6 p.i. using Western blot (bottom). The 6 day p.i. shEcad#21 and 4 and 6 day p.i. shEcad#23 samples are not shown because they did not meet our qPCR normalization criteria. The generally poor PLoS ONE | www.plosone.org 10 October 2010 | Volume 5 | Issue 10 | e13665