Inhibition of EGFR Localization to Mitochondria Figure 3. Inhibition of EGFR by hUCBSC treatment. For in vitro experiments, glioma cells (U251, U87 and 5310) were co-cultured with hUCBSC for 72 hours. For in vivo experiments, U251 (16106 cells) and 5310 (86105 cells) tumor cells were intracerebrally injected into the right side of the brain of the nude mice. Nude mice with pre-established intracranial human glioma tumors (U251 or 5310) were treated with 26105 hUCBSC by intracranial injection. Seven days after tumor implantation, the mice were injected with hUCBSC towards the left side of the brain. Fourteen days after hUCBSC administration, the brains were harvested, sectioned, and stained with appropriate antibodies. (A) U87, U251 and 5310 glioma cells alone and after co-culture with hUCBSC were labeled with mouse anti-EGFR antibody and processed for immunofluorescence. EGFR was conjugated with Alexa Fluor 488 (green) secondary antibody (n = 3; bar = 100 mm). (B) Control and hUCBSC-treated tumor sections were labeled with mouse anti-EGFR antibody and processed for DAB immunohistochemistry. For (C) and (E), total mRNA was extracted, converted to cDNA, and subjected to semi- quantitative RT-PCR. (C) Control and hUCBSC-treated U251, U87 and 5310 in vitro cDNAs were subjected to RT-PCR using EGFR, FAK and c-Src primers. (D) Quantitative analysis of (C). *Significant at p,0.05 compared to respective control glioma cells. **Significant at p,0.01 compared to respective control glioma cells. (E) Control and hUCBSC-treated U251 and 5310 in vivo cDNAs were subjected to RT-PCR using EGFR, FAK and c-Src primers. b-actin served as a loading control. (F) Quantitative analysis of (E). *Significant at p,0.05 compared to respective control glioma tissues. For (G) and (I), 40 mg of proteins were loaded onto 8% gels and transferred onto nitrocellulose membranes, probed with respective antibodies, and developed by autoradiography. (G) Control and hUCBSC-treated U251, U87 and 5310 glioma cell lysates were processed and immunoblotted with EGFR, FAK and c-Src antibodies. (H) Quantitative analysis of (G). **Significant at p,0.01, *Significant at p,0.05 compared to respective control glioma cells. (I) Control and hUCBSC-treated U251 and 5310 in vivo brain tissue lysates were processed by standard Western blotting and probed with EGFR, FAK and c-Src antibodies. GAPDH served as a loading control. (J) Quantitative analysis of (I). **Significant at p,0.01, *Significant at p,0.05 compared to respective control glioma tissue lysates. For (C–J), n$3. doi:10.1371/journal.pone.0031884.g003 PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e31884