Boyden chamber assay Invasion of breast cancer cells was examined using 24-well Boyden chambers (Costar, Corning, NY, USA) with 8-mM inserts coated with fibronectin (Roche, Mannheim, Germany) and matrigel (BD Biosciences, USA). The lower chamber was filled with L-15/BSA medium containing 10 ng/ml SDF. MDA-MB- 436 cells (56104 cells/well) were pretreated with mitomycin (10 mg/ml) for 2 h to block proliferation and then were plated on the inserts with, or without HGF/SF and cultured at 37uC in the upper chambers. After 8 hours, invading cells that crossed the inserts were stained with viola crystalline (0.005%, Sigma, St. Louis, USA) and were counted as cells per field of view using phase-contrast microscopy. Flow cytometry of membrane and intracellular CXCR4 expression Membrane CXCR4 expression was determined by flow cytometry (BD, CA, USA) as described previously [11] using anti-human CXCR4 monclonal antibody (clone 12G5) (Santa Cruz, CA, USA), followed by secondary FITC-conjugated donkey anti-mouse IgG. Isotype matched IgG2a was used as control, showing background labeling with no differences between the treatments. Ratios of mean values of Ab labeled cells to secondary IgG only control samples were calculated using Cell Quest software (BD, CA, USA), and the results were expressed in arbitrary units (AU). Flow cytometry analysis with additional anti CXCR4 mAbs, clones 6H8 and 1D9, demonstrated similar to 12G5 increase following HGF treatment. For the analysis of intracellular CXCR4 levels, MDA-MB-436 cells were immunola- beled with anti-human CXCR4 mAb after blocking the membrane receptors by a 1-hour incubation with 10 mg/ml non-conjugated 12G5 mAb. Membrane CXCR4 internalization was assayed as previously described: MDA-MB-436 cells were pre- incubated for 30 minutes at 4uC with 10 mg/ml of anti human CXCR4-PE Ab, transferred to 37uC for the indicated times, and then washed for 2 minutes on ice at acidic (pH2.2) buffer to remove the surface-bound Ab. The relative proportion (%) of internalized receptor (labeling after acid wash) with respect to total fluorescence intensity was determined by flow cytometry. CXCR4 and protein kinase C f translocation assay Cells were cultured for 48 hours before the experiment. They were then starved in binding medium for 3 hours, followed by stimulation with 50 ng/ml HGF at 37uC for 10 minutes before fixation with 4% formaldehyde. As the inhibitor control, cells were treated with 50 mM NSC23766 (Santa Cruz, CA, USA) at 37uC for 1 hour before HGF stimulation. Cells were then permeabilized with 0.2% Triton X-100 in buffer (10 mM HEPES, 20 mM KH2PO4, 5 mM EGTA, 2 mM MgCl2, PBS, pH 6.8), stained with polyclonal antibodies against various PKC isotypes and probed with FITC-labeled goat anti-rabbit antibody. CXCR4 was labeled with 10 mg/ml anti-human CXCR4 mAb. Cells were visualized using a Zeiss LSM 710 inverted fluorescent confocal microscope (Carl Zeiss, Inc, Germany). qRT-PCR qRT-PCR was performed using an ABI Prism 7000 System (Applied Biosystems, Waltham, MA, USA). SYBR Green was used to detect the PCR products. All reactions were performed in triplicate in a 20-ml reaction volume. Primers for human CXCR4, MT1-MMP, GAPDH, HPRT and mouse 18S rRNA were obtained from Takara (Table S3). PCR amplification consisted of an initial denaturation step at 95uC for 5 min, followed by 40 PKCf Induced by HGF Increases CXCR4 Expression cycles of PCR at 95uC for 20 s, 60uC for 30 s. Standard curves were generated, and the relative amount of target gene mRNA was normalized to GAPDH mRNA. Specificity was verified by melt curve analysis and agarose gel electrophoresis. To quantify cancer metastasis in mouse lungs and livers, qRT-PCR for human hypoxanthine-guanine phosphoribosyltransferase (hHPRT) was performed on TRIzol-isolated total RNA using the primers described for hHPRT and mouse 18S rRNA. After reverse transcription for 45 min at 42uC and Taq activation for 3 min at 95uC, 40 cycles of PCR at 95uC for 12 s and 60uC for 15 s were performed. The relative amount of hHPRT mRNA was normalized to mouse 18S rRNA. Transfections Transient transfections and siRNA-mediated protein silencing in MDA-MB-436 cells were performed using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. siRNAs were chemically synthesized as double-stranded RNA (GenePharma Co., Ltd., Shanghai, China). siRNAs against canine PKCf [23]or Rac1 [58] have been described as follows: PKCf-siRNA 1 sense: 59-GGCUGUUC- CUGGUCAUAGA-39; antisense: 59-UCUAUGACCAGGAA- CAGCC-39. PKCf-siRNA 2 sense: 59-GGUGCACACUUUC- CACAGA-39; antisense: 59-UCUGUGGAAAGUGUGCACC-39. Rac1-siRNA sense: 59-GAGGCCUCAAGACAGUGUUU- GACGA-39; Rac1-siRNA antisense: 59-UCGUCAAACACUGU- CUUGAGGCCUC-39. GFP-siRNA (GenePharma Co., Ltd., Shanghai, China) was used as negative control. Western blotting assay Western blotting was carried out as described previously [59]. Briefly, protein extracts were resolved by 10% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against human CXCR4 (Abcam, Cambridge, MA, USA), phosporylated CXCR4 (p-CXCR4) (Abcam, Cambridge, MA, USA), PKCf (Bioworld, Louis Park, MN, USA), phosporylated PKCf (p-PKCf) (Bioworld, Louis Park, MN, USA), Akt/PKB (Santa Cruz, CA, USA), phosporylated Akt/PKB (Santa Cruz, CA, USA), Rac1 (Santa Cruz, CA, USA), Met (Bioworld, Louis Park, MN, USA), phosporylated Met (p-Met) (Bioworld, Louis Park, MN, USA), MT1-MMP(Abcam, Cambridge, MA, USA) and visualized by an enhanced chemiluminescence assay (Pierce). Rac activation assay Rac1 activity was measured using the rac1 activation assay kit (Upstate Biotechnology, NY, USA) with slight modification. Briefly, whole-protein extracts were immunoprecipitated with protein binding domain of p21 activation kinase-1 (PAK1-PBD). PAK1-PBD only binds to activated forms of rac1 and cdc42. Immunoprecipitated proteins were separated in SDS-PAGE (10%) and blotted with anti-rac1 (Santa Cruz BiotechnologyInc., CA, USA). Immunohistochemistry For immunohistochemistry analysis, anti-p-PKCf monoclonal antibody or anti-p-CXCR4 polyclonal antibody was used as the primary antibody for overnight incubation at 4uC. The sections were subsequently treated with secondary antibody, followed by further incubation with the streptavidin-horseradish peroxidase complex. Diaminobenzidine was used as a chromogen, and sections were lightly counterstained with hematoxylin. The percentage of p-PKCf- and p-CXCR4-positive tumor cells was calculated by counting 1,000 tumor cells. PLoS ONE | www.plosone.org 13 January 2012 | Volume 7 | Issue 1 | e29124