Inhibition of EGFR Localization to Mitochondria Figure 4. Inhibition of EGFR and FAK co-localization by hUCBSC. (A) Control and hUCBSC-treated U87, U251 and 5310 glioma cells were labeled with Ki67 antibody and processed for immunofluorescence. Ki67 was conjugated with Alexa Fluor 594 (red) secondary antibody (n$3; bar = 100 mm). (B) Control and hUCBSC-treated U87, U251 and 5310 glioma cells were labeled with pEGFR and pFAK antibodies and processed for immunofluorescence. pFAK was conjugated with Alexa Fluor 488 (green) and pEGFR was conjugated with Alexa Fluor 594 (red) secondary antibodies (n$3; bar = 100 mm). (C) Control, EGF-treated, EGF+hUCBSC-treated, and hUCBSC-treated U251, U87 and 5310 cells were processed and 40 mg of proteins were loaded onto 8% gels and transferred onto nitrocellulose membranes, probed with EGFR and FAK antibodies, and developed by autoradiography. GAPDH served as a loading control. n = 3. (D) Quantitative analysis of (C). *Significant at p,0.05 compared to respective control glioma cells by one-way ANOVA. doi:10.1371/journal.pone.0031884.g004 results, we ran Western immunoblots of pEGFR, pFAK and p-c- Src in whole cell lysates and whole tissue lysates of control and hUCBSC-treated glioma samples. We observed downregulation of phosphorylated forms of EGFR, FAK and c-Src in hUCBSC- treated glioma cells (Fig. 7A). Downregulation of active forms of EGFR and c-Src are more significant compared to FAK in these cell lysates (Fig. 7B). In a similar fashion, these active forms were also downregulated in hUCBSC-treated total tissue lysates of brain xenografts (Figs. 7C, 7D). Compared to total tissue lysates, the mitochondrial fractions of brain xenografts showed high concen- tration of phospho-forms of EGFR, FAK and c-Src (Figs. 7E, 7F). The mitochondrial fractions of U251 control tissues show 68.4960.42% (mean 6 S.D.) of pEGFR whereas 5310 control tissues show about 53.7262.21% of pEGFR, which means that out of total pEGFR present in these glioma tissues, major portion of activated EGFR is translocated to mitochondria (Fig. 7E). The mitochondrial fractions of hUCBSC treated U251 tissues show 33.2161.52% of pEGFR whereas hUCBSC treated 5310 tissues show about 24.3960.53% of pEGFR, which means that hUCBSC inhibited the translocation of pEGFR to mitochondria. Since, endosomal-lysosomal internalization upon translocation of acti- vated plasma membrane bound EGFR takes place, we tested this pathway by immunohistochemistry methods. Probing the control and hUCBSC-treated nude mice brain sections with Cathepsin-L antibody, we observed that Cathepsin-L is highly activated in U251 and 5310 xenograft tumors, and the expression is highly PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e31884