Inhibition of EGFR Localization to Mitochondria Figure 1. Expression of EGFR in GBM-patient derived specimens. (A) Control brain and GBM-patient derived specimens were subjected to DAB immunohistochemistry using anti-rabbit EGFR antibody (bar = 200 mm). (B) mRNA extracted from the above specimens was subjected to RT-PCR using primers specific for EGFR, FAK, c-Src and b-actin (loading control). (C) Quantitative analysis of (B). (D) Tissue lysates (40 mg protein) of the above specimens were subjected to Western blotting using the following antibodies: EGFR, pEGFR, FAK, pFAK, c-Src and phospho-c-Src. Mouse anti-GAPDH (1:1000) served as the loading control. (E) Quantitative analysis of (D). *Significant at p,0.05 compared to control brain samples. doi:10.1371/journal.pone.0031884.g001 inhibited by hUCBSC treatments (Fig. 6A). Similarly, in vivo samples also show inhibition of translocation of pEGFR to mitochondria (Fig. 6B). Thus, under both in vitro and in vivo conditions, hUCBSC prevented the translocation of pEGFR to mitochondria. As mentioned earlier, activation of EGFR stimu- lates activation of many different proteins like FAK and c-Src. We therefore evaluated the expression of phospho-c-Src in mitochon- dria of glioma cells. All of the glioma cell lines in the present study showed higher expression of phospho-c-Src (Fig. 6C). We also observed higher expression of phospho-c-Src in vivo in nude mice brain tumors (Fig. 6D). In both cases, the expression of phospho-c- Src was inhibited by hUCBSC treatments. To confirm these PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31884