PKCf Induced by HGF Increases CXCR4 Expression Figure 1. Immunohistochemical staining of HGF, c-Met and CXCR4 in breast cancer specimens. Original magnification, 4006. (A) Representative micrographs of immunohistochemical results for negative HGF, c-Met or CXCR4 staining in breast benign diseases (an atypical hyperplasia or carcinoma in situ) and for positive HGF, c-Met or CXCR4 staining in an invasive breast carcinoma; (B) Representative micrographs of immunohistochemical results for positive HGF, c-Met and CXCR4 staining cell counts, where the intensity of immunohistochemical staining correlates with the histopathological grading of invasive breast carcinomas. doi:10.1371/journal.pone.0029124.g001 tively examined in MDA-MB-436 cells incubated for 24 hours with HGF. While cell surface (membrane) expression of the receptor was upregulated by 2.5-fold, only a 1.4-fold increase was found for the level of intracellular CXCR4 (Figure 2F). Further- more, a time course analysis of surface-bound anti-CXCR4 mAb internalization showed that CXCR4 receptor endocytosis was reduced by 2-fold in HGF-stimulated cells compared with untreated counterparts (Figure 2G), suggesting that in the absence of ligand, HGF stimulation may contribute to longer duration for CXCR4 to be detectable on cell membrane. PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e29124