the cell [47–49]. Protein kinases, protein phosphatases, and even transcriptional factors, which are not conventionally mitochondri- al resident members, are revealed to have a mitochondrial location [44,48]. However, the molecular mechanisms underlying EGFR localization in mitochondria remain largely unknown. EGFR mitochondrial localization is regulated by either autophagy or programmed cell death and is correlated with cell survival [50]. Demory et al. [15] postulated that EGF stimulation would lead to EGFR and c-Src activation followed by translocation of these molecules to the mitochondria, phosphorylation of CoxII by EGFR and/or c-Src, reduction in oxidative ATP and free radical production, and an increase in cell viability. Mitochondrial- localized EGFR is independent of its internalization and may be correlated with cell survival and participate in the ligand-induced programmed cell death [20]. In our studies, we observed that phosphorylation of EGFR is inhibited by hUCBSC treatments. Unless EGFR is activated, it cannot translocate to mitochondria or any other sub-cellular organelles. Hence, we could not observe translocation of EGFR to mitochondria in hUCBSC-treated in vitro and in vivo samples. Further, we did not observe any co- localization of activated EGFR and FAK molecules in mitochon- dria after hUCBSC treatments. In summary, our data indicate that hUCBSC coordinately regulates EGFR signaling at cytosolic and mitochondrial levels in glioma cells. The effect of hUCBSC action is to inhibit cell cycle progression and reduce cell growth and viability as reported previously. The reported down-regulation of EGFR in GBM tumors, together with the ability to regulate oncogenic EGFR signaling in glioma cell lines, suggests the therapeutic potential of hUCBSC in regulating GBM tumors. The inhibition of EGFR by hUCBSC can be effective in the treatment of GBM. Our previous findings indicated that hUCBSC can inhibit cell viability by initially reducing the content of mitochondrial membrane potential [21]. The results of the present study show that inhibition of EGFR localization to mitochondria provide further evidence of the growth inhibitory effect of hUCBSC on glioma cells. Our findings show that hUCBSC inhibit EGFR localization in mitochondria in GBM cells and provide relevant preclinical information that can be exploited for the development of effective hUCBSC-based therapies. Materials and Methods Ethics Statement After obtaining informed consent, human umbilical cord blood was collected from healthy volunteers according to a protocol approved by the Peoria Institutional Review Board, Peoria, IL, USA. The consent was written and approved. The approved protocol number is 06-014, dated December 10, 2009 and renewed on November 11, 2010. 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. The consent was written and approved. The approved protocol number is 851, dated November 20, 2009 and renewed on March 15, 2011. Cell cultures Two high-grade human glioma cell lines (U251, U87) and one xenograft cell line (5310) were used for this study. U251 and U87 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). The 5310 xenograft cell line was kindly provided by Dr. David James at University of California, San Francisco. U251 and U87 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) (Hyclone, Inhibition of EGFR Localization to Mitochondria Logan, UT) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA). The 5310 xenograft cell line was grown in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin- streptomycin. For hUCBSC, after obtaining informed consent, human umbilical cord blood was collected from healthy volunteers according to a protocol approved by the University Of Illinois College Of Medicine at Peoria Institutional Review Board. Cord blood was processed as described previously by sequential Ficoll density gradient purification [51]. Next, we selected cells using CD29+ and CD81+ markers as described previously. The nucleated cells were grown in mesencult medium (Stem Cell Technologies, Vancouver, Canada) supplemented with 20% FBS and 1% penicillin-streptomycin and plated in 100-mm culture dishes. All the cells were grown at 37uC in an incubator with a 5% CO2 atmosphere. For co-culture experiments, hUCBSC and glioma cells were cultured at a ratio of 1:1 (16106 for each cell line) for 72 hours. Co-cultures of hUCBSC and U251, hUCBSC and U87 were grown in DMEM (with 10% FBS) and co-cultures of hUCBSC and 5310 were grown in RPMI-1640 (with 10% FBS) and then FACS sorted as described previously [24]. Collection of patients’ specimens GBM specimens were collected from patients with histologic diagnosis of primary GBM (WHO grade IV astrocytoma) in accordance with the protocol approved by the University Of Illinois College Of Medicine at Peoria Institutional Review Board. Tumor diagnosis and grading were blind-reviewed by a neuropa- thologist. Samples that did not satisfy the inclusion criteria of primary GBM (e.g., GBM with oligodendroglioma features) were excluded from the study. BrdU assay for cell proliferation Cell proliferation analysis was performed using Cell Prolifera- tion ELISA (colorimetric) BrdU incorporation assay (Roche diagnostics, Indianapolis, IN) according to the manufacturer’s protocol. Cells (0.56104 cells/well) were cultured in 96-well plates for different time periods (48 h, 72 h and 96 h for coculture treatments with hUCBSC) and treatments and incubated for 12 hours with BrdU reagent. An anti-BrdU antibody was added, and the immune complexes were detected by subsequent substrate reaction. The reaction product was quantified as per manufac- turer’s instructions. EGF and TMZ treatments Glioma cells were subjected to serum starvation for 3 hours and then treated with EGF (100 ng/mL) for 1 hour in serum free medium. After EGF treatment, the cells were washed twice with PBS and harvested. In another experiment, glioma cells that were given EGF treatment were co-cultured with hUCBSC in 1:1 ratio for 72 hours as described above. For Temozolomide (TMZ) treatments, the glioma cells were subjected to serum starvation for 3 hours and then treated with TMZ (1000 mM) for 24 h in serum free medium. In combination treatments with hUCBSC, stem cell treatments were given at different time periods (48 h, 72 h and 96 h) either before or after treatment with TMZ. In both EGF and TMZ treatments, we tested different concentrations and time periods and finally selected the above time points and concentra- tions which yield optimum results (data not shown). RNA analysis Total RNA from GBM patient-derived tumor specimens (frozen) and from control and hUCBSC-treated glioma cell lines was extracted using the RNeasy Mini Kits (Qiagen, Valencia, CA). PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e31884