PKCf Induced by HGF Increases CXCR4 Expression Figure 5. HGF results in PI3K/Akt pathway phosphorylation and activates CXCR4 phosphorylation via PKCf. (A). Detection of phosphorylated Akt or of the respective total Akt protein expression and Rac1-GTP or total Rac1 expression by western blot analysis. MDA-MB-436 cells were added with PBS or were exposed to HGF/SF (for 10 minutes at 50 ng/ml) after preincubation with the PI3-kinase inhibitors wortmannin (at 100 nM, for 60 min) or LY 294002 (at 10 mM, for 60 min) as indicated. The experiment was repeated twice with similar results. A representative study is shown. (B) Membrane CXCR4 expression in MDA-MB-436 cells cultured in the absence or presence of wortmannin (100 nM) or LY 294002 (50 mM, starting 1 hour prior to HGF/SF treatment) with or without HGF/SF treatment (for 16 hour at 50 ng/ml) as indicated. The flow cytometry analysis data are shown in arbitrary units (AU) as the mean 6 SD of three independent experiments. # P,0.01 as compared to PBS. (C) MDA-MB-436 cells were exposed to HGF with or without PI 3-kinase inhibitor LY294002 (30 mM) or Akt inhibitor III (50 mM) for various amounts of time, which resulted in the phosphorylation of PKCf. Western blotting of total protein were performed in triplicate. A representative study is shown. (D). Cellular distribution of PKCf and CXCR4 before and after 30 minutes of HGF stimulation. HGF, 50 ng/ml, for 10 minutes; LY294002, 30 mM, for 60 min. The experiment was repeated three times with similar results. A representative study is shown. doi:10.1371/journal.pone.0029124.g005 adhesion and migration by regulating Rho and PKCf [57]. In order to investigate whether PI3K/Akt were involved in the effects of HGF-induced CXCR4 expression in MDA-MB 436 cells, we examined the changes of CXCR4 expression after HGF-induced PKCf activation by PI3K/Akt inhibitors. Western blot assays and immunofluorescent staining data suggested that PI 3K/Akt are required for PKCf activation and CXCR4 expression. Further- more, Rac1 activation is also dependent on PI3-kinase activity. The lack of complete inhibition may indicate that activation of a small percentage of Akt/PKB is sufficient for chemotaxis, or that an unidentified signaling molecule downstream of the PI 3K shares a partially redundant function with Akt/PKB. Those results also indicated that the mechanism of HGF-induced CXCR4 expression in the MDA-MB-436 breast cancer cell line involved a pathway of activation of the PKCf and PI 3K/Akt, and that signals stimulated by HGF passed on from PI 3K/Akt to Rac1 and PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e29124