cDNA was obtained by using Transcriptor first-strand cDNA synthesis kit (Roche, Indianapolis, IN) by reverse transcription- based PCR (RT-PCR). All cDNAs were normalized to the b-actin RT-PCR product. PCR amplification was performed using the primer sets listed below, amplified by 35 cycles of PCR (94uC, 1 min; 60uC, 1 min; 72uC, 1 min) using 20 pM of specific primers. Primers used for RT-PCR EGFR Forward 59ccaagggagtttgtggagaa39 Reverse 59cttccagaccagggtgttgt39 FAK Forward 59ggtgcaatggagcgagtatt39 Reverse 59gccagtgaacctcctctga39 c-Src Forward 59gggtagcaacaagagcaa39 Reverse 59gagttgaagcctccgaacag39 b-Actin Forward 59ggcatcctcaccctgaagta39 Reverse 59ggggtgttgaaggtctcaaa39 Immunoblot analysis Single and co-cultures of glioma cells or nude mice brain tissues (frozen) were harvested and homogenized in four volumes of homogenization buffer as described previously [51]. Samples (40– 50 mg of total protein/well) were subjected to 8–12% SDS-PAGE and transferred onto nitrocellulose membranes. The reaction was detected using Hyperfilm-MP autoradiography film (Amersham, Piscataway, NJ). The following antibodies were used for Western blot analysis: mouse anti-EGFR (1:500), goat anti-pEGFR (1:500), mouse anti-FAK (1:500), goat anti-phospho-FAK (1:500), mouse anti-c-Src (1:500), mouse anti-MEK1 (1:500), rabbit anti-RAF1 (1:500), mouse anti-STAT3 (1:500), mouse anti-PI3K (1:500) (all from Santa Cruz Biotechnology, Santa Cruz, CA), and rabbit anti- phospho-c-Src (1:1000; Cell Signaling). Immunoreactive bands were visualized using chemiluminescence ECL Western blotting detection reagents (Amersham). Immunoblots were stripped and redeveloped with GAPDH antibody [mouse anti-GAPDH (1:1000; Santa Cruz)] to ensure equal loading levels. Experiments were performed in triplicate. The bands in each blot were quantified using ImageJ software and the values were expressed in relation of GAPDH values. Intracranial tumor growth inhibition The Institutional Animal Care and Use Committee of the University Of Illinois College Of Medicine at Peoria approved all surgical interventions and post-operative animal care. For the intracerebral tumor model, U251 (16106 cells) and 5310 (86105 cells) tumor cells were intracerebrally injected into the right side of the brain of the nude mice as described previously [23]. Seven days after tumor implantation, the mice were injected with hUCBSC in the contralateral left brain hemisphere. The ratio of the hUCBSC to cancer cells was maintained at 1:4. Three weeks after tumor inoculation, six mice from each group were sacrificed by cardiac perfusion with 4% formaldehyde in PBS, their brains were removed, and paraffin sections were prepared. Sections were stained with H&E to visualize tumor cells and to examine tumor volume. The sections were blindly reviewed and scored semi- quantitatively for tumor size. Whole-mount images of brains were also taken to determine infiltrative tumor morphology. The average tumor area per section integrated to the number of sections where the tumor was visible was used to calculate tumor volume and compared between controls and treated groups. Immunoblot analyses and RT-PCR were carried out on fresh brain tissues. Inhibition of EGFR Localization to Mitochondria Immunostaining analyses GBM patient-derived specimens and brains of control and hUCBSC-treated mice were fixed in formaldehyde and embed- ded in paraffin as per standard protocols. Sections were deparaffinized and blocked in 1% BSA in PBS for 1 hour, and the sections were subsequently transferred to primary antibody diluted in either 1% BSA (for pEGFR and pFAK antibodies) or 10% normal goat serum (1:100) (for all other antibodies). Sections were allowed to incubate in the primary antibody solution overnight at 4uC in a humidified chamber. Sections were then washed in PBS and placed in either 1% BSA or 10% goat serum with the appropriate secondary antibody. The sections were incubated with the secondary antibody for 1 hour and visualized using a confocal microscope. Transmitted light images were obtained after H&E staining as per standard protocol to visualize the morphology of the sections. Negative controls were maintained without primary antibody or by using IgG fraction. For DAB immunohistochemistry, sections were probed with antibodies and then stained with DAB (Sigma) and further stained with Hematoxylin. For immunofluorescence studies, cultured glioma cells plated in 2-well chamber slides were rinsed twice with phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde. After additional PBS rinses, cells were blocked with 16 PBS with 1% bovine serum albumin (BSA) for 1 hour. Primary antibodies (1:100 dilutions) were diluted in either 10% goat serum or 1% BSA and applied overnight at 4uC. Alexa Fluor-conjugated secondary antibodies were diluted (1:200) in either 10% goat serum or 1% BSA and applied individually for 1 hour at room temperature. We used the following antibodies: mouse anti-EGFR, mouse anti-Ki67, rabbit anti-FAK, goat anti- pEGFR, mouse anti-phospho-c-Src (all from Santa Cruz Biotechnology). For mitochondrial localization, we used Mito Tracker Green (Invitrogen) as per the manufacturer’s instruc- tions. Before mounting, the cells were stained with 49, 6- diamidino-2-phenylindole (DAPI). The cells were observed using a fluorescence microscope (Olympus IX71, Olympus, Melville, NY) and/or a confocal microscope (Olympus Fluoview) and photographed. We used same antibodies for both immunofluo- rescence studies and immunohistochemistry studies. Negative controls were maintained without primary antibody or by using IgG fraction. Statistical analyses Values are shown as mean 6 SD of at least three independent experiments. Results were analyzed using a two-tailed Student’s t- test using Graph Pad Prism version 3.02, a statistical software package. Results were considered statistically significant at p,0.05 or p,0.01. For comparing more than two groups, statistical significance using one way analysis of variance (ANOVA) was used. Data for each treatment group were represented as mean 6 SEM and compared with other groups for significance by one-way ANOVA followed by Bonferroni’s post hoc test (multiple comparison tests) using Graph Pad Prism version 3.02. Results were considered statistically significant at a p,0.05 or p,0.01. Supporting Information Figure S1 Inhibition of lysosomal internalization by hUCBSC treatments. Control and hUCBSC-treated nude mice brain sections were immunoprobed with mouse anti- Cathepsin L antibody and processed for DAB staining followed by Hematoxylin staining. Bar = 200 mm. n$3. (TIF) PLoS ONE | www.plosone.org 12 February 2012 | Volume 7 | Issue 2 | e31884