laboratory. All injected tumors formed a dominant mass of several millimeters in the cerebral hemisphere ipsilateral to the injection, up to a maximum diameter of approximately 6 mm in the moribund state. The degree of invasiveness was evaluated on the H&E sections by an observer (C.G.) who was blind to tumor E- cadherin status. In tumors which were classified as ‘‘highly invasive’’, tumor cells could be easily identified extending along the commissural structures (corpus callosum and anterior commissure) to the opposite hemisphere. In tumors which were considered ‘‘minimally invasive’’ or ‘‘non invasive’’ a ‘‘nearly sharp’’ border could be identified between the tumor mass and surrounding brain parenchyma, with minimal or no intermixing of tumor cells with surrounding parenchyma at the edge of the tumor (Figure S2). A number of xenografts showed intermediate features (‘‘moderate invasiveness’’) with cells extending well away from the main tumor mass, but largely remaining in the ipsilateral hemisphere. Conventional cell culture Established conventional cell lines were cultured on tissue- culture treated plastic dishes at 37uC, 5% CO2, in DMEM media containing 10% Fetal Bovine Serum (not heat-inactivated), an additional 2 mM L-glutamine, and 1% non-essential amino acids. Glioma cell lines include A172, CCF, D32, D37, H4, Hs683, SW1088, SW1783, TP265, TP365, TP483, U87, U251, and U373 (obtained from Bob Jenkins, Mayo Clinic, Rochester, MN); G112P, SF767, T98G, and U118 (obtained from Joe Loftus, Mayo Clinic, Scottsdale, AZ); LN229 (obtained from Wei Zhang, MD Anderson Cancer Center, Houston, TX); and SF767 (obtained from the UCSF/Neurosurgery Tissue Bank, San Francisco, CA). MCF7, MDA-MB-231, and UMRC3 cell lines were also used. Constructs The MISSION Non-Target shRNA control vector pLKO-non target (SHC002) and pLenti-human E-cadherin shRNA vectors, both expressing a puromycin resistance gene, were purchased from the Mission RNAi Consortium shRNA collection (Sigma-Aldrich, St. Louis, MO) and obtained from the Mayo Clinic Comprehensive Cancer Center RNA Interference Techonology Resource. E- cadherin shRNA product numbers are: pLKO-shEcad#20, TRCN0000039663; pLKO-shEcad#21, TRCN0000039664; pLKO-shEcad#22, TRCN0000039665; pLKO-shEcad#23, TRCN0000039666; pLKO-shEcad#24, TRCN0000039667. Virus production and infections Lentivirus stocks were produced using VirapowerTM lentivirus packaging mix and the 293FT cell line according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). SF767 cells (obtained from J. Loftus) grown to 50% confluence were incubated for 24 hours in a 1:1 dilution of virus:media with 4 mg/ml Polybrene. After a 24-h recovery in complete media without virus, polyclonal stable cell lines were selected and maintained in media containing 5 mg/ml puromycin. In the parallel SF767 and MCF7 infection experiment, a 1:4 dilution of virus:media with 6 mg/ml Polybrene was used. Preparation of whole cell lysates for western blot Flank xenograft tissue lysates: Flash frozen flank xenograft tissues were homogenized at 0uC in SDS Lysis buffer (2% w/v SDS, 4 M deionized urea, 62.5 mM Tris-Cl pH 6.8, 1 mM EDTA, 5% v/v b-Mercaptoethanol, with 1 mM Na3VO4, 50 mM NaF, and 1 mM PMSF). Lysates were then cleared by centrifugation at 13000 rpm at 4uC for 20 minutes. Total protein E-Cadherin Expression in GBM in the supernatents was quantified using the nitric acid method [59], while the remaining lysates were mixed with Laemmli Sample Buffer (2X final concentration, 0.1 M Tris-Cl pH 6.8, 2% SDS, 10% sucrose, 0.24 M b-Mercaptoethanol, 0.008% bromo- phenol blue) and boiled for 5 minutes before being analyzed by Western Blot. Conventional cell culture lysates: Whole cell lysates of conventionally cultured cell lines were made by lysing the cells in ice cold RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% Igepal CA-630 (NP-40 substitute), 0.5% Deoxycholic Acid, 0.1% SDS, with 1 mM Na3VO4, 1 mM EDTA, 50 mM NaF, 1 mM PMSF, 5 mg/ml leupeptin, and 2 mg/ml aprotinin) for 7 minutes, followed by brief sonication. Ten ml samples of each lysate were quantitated for total protein using the BioRad Protein Assay Dye Reagent (BioRad Laboratories, Hercules, CA). Remaining lysates were mixed with Laemmli Sample Buffer (2X final concentration) and boiled for 5 minutes before being analyzed by Western Blot. Western blotting Equal mg amounts of protein lysates were separated by SDS- PAGE and transferred to nitrocellulose filters using standard methods. Blots were blocked in 5% nonfat dry milk in Tris- buffered saline (TBS), pH 7.4 before being incubated in primary antibodies in 5% milk/TBS. Blots were rinsed three times and washed four times 5 minutes in TBS +0.1% Tween 20 (TBST). Blots were then incubated with HRP-conjugated secondary antibodies in 5% milk/TBS, and rinsed and washed in TBST as before. Proteins were detected using Amersham ECL Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, United Kingdom). Primary antibodies were as follows: mouse anti- p120 catenin (clone 15D2, Zymed/Invitrogen), rabbit anti-b catenin (C2206, Sigma-Aldrich), mouse anti-E-cadherin (clone 36, BD Biosciences, San Jose, CA), mouse anti-N-cadherin (clone 3B9, Zymed/Invitrogen), mouse anti-cadherin-11 (clone 5B2H5, Zymed/Invitrogen), rabbit anti-actin (A2066, Sigma-Aldrich), and mouse anti-a-tubulin (clone B-5-1-2, T5168, Sigma-Aldrich). HRP-conjugated secondary antibodies (anti-mouse and anti- rabbit) were obtained from Jackson ImmunoResearch Laborato- ries, West Grove, PA. Immunofluorescence SF767 and MCF7 cells were plated on glass coverslips and allowed to adhere for a minimum of 24 hours. For combined E- cadherin and actin analysis, cells were fixed with 3% paraformal- dehyde at room temperature for 30 minutes, then washed twice in PBS+10 mM glycine. Cells were permeabilized with PBS/0.2% Triton X-100 for 2.5 minutes at room temperature, then washed again with PBS/glycine before blocking and applying mouse anti- E-cadherin primary antibody (clone 36, BD Biosciences). For combined E-cadherin and b-catenin analysis, cells were fixed/ permeabilized by incubation in 100% methanol at 220uC for 7 minutes, then washed with PBS before blocking and applying mouse anti-E-cadherin and rabbit anti-b catenin (C2206, Sigma- Aldrich) primary antibodies. Secondary antibodies were goat anti mouse Alexa 488 and goat anti rabbit Alexa 594 (Invitrogen). Actin was localized by incubation with Alexa 594-conjugated phalloidin (Invitrogen). SF767 and MCF7 cells were stained simultaneously using identical conditions. Coverslips were mount- ed on glass slides with Aqua Poly/Mount (Polysciences, Inc. Warrington, PA). Cells were visualized with a Zeiss LSM 510 META laser scanning confocal microscope (Carl Zeiss Microima- ging, Heidelberg, Germany) using a Plan-Apochromat 63x/1.4 oil immersion objective. Images were acquired with the Zeiss AIM PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13665