PKCf Induced by HGF Increases CXCR4 Expression Figure 2. HGF upregulates CXCR4 expression and membrane presentation in human breast cancer cells. (A). qRT-PCR analysis of the total CXCR4 mRNA extracted from HGF- or SDF-1-treated MDA-MB-436 and MCF-7 cells. Results are presented as the mean 6 SD of three independent experiments. # P,0.01 as compared to PBS. (B). Western blot analysis of the total protein expression and phosphorylation levels of Met or CXCR4 in MDA-MB-436 and MCF-7 cells cultured for 24 hours with and without the presence of 50 ng/ml HGF or 20 ng/ml SDF-1. The experiment was repeated twice with similar results. A representative study is shown. (C). Time course of the relative extracted CXCR4 mRNA expression in MDA- MB-436 and MCF-7 cells following stimulation with 50 ng/ml HGF. Results are presented as the mean 6 SD of three independent experiments. # P,0.01,* P,0.05 as compared to PBS. (D). Western blot analyses of CXCR4 protein expression in HGF-treated MDA-MB-436 and MCF-7 cells. The experiment was repeated three times with similar results. A representative study is shown. (E). Flow cytometric analysis of MDA-MB-436 cells stained for the expression of CXCR4 using a CXCR4 monoclonal antibody and isotype controls. The experiment was repeated three times with similar results. A representative study is shown. (F). Increased membrane and intracellular CXCR4 labeling in MDA-MB-436 cells incubated for 24 hours in the presence of 50 ng/ml HGF compared with untreated cells (PBS) taken as 1. Flow cytometry analysis data (mean6SD of three independent experiments) are shown. # P,0.01,* P,0.05 as compared to PBS. (G). Decreased internalization rate of anti-CXCR4-PE mAb in MDA-MB-436 cells treated for 24 hours with HGF compared with PBS. Results are presented as the mean 6 SD of three independent experiments. * P,0.05 as compared to PBS at each time point. doi:10.1371/journal.pone.0029124.g002 PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e29124