Proliferation and Cell viability assay MCF-7 cells and MDA-MB 436 cells (36105/100 ml) were cultured in serum-free medium with HGF (50 ng/ml) and/or PS-f peptides (10 mM), AMD3100 (1 mM), NSC23766 (25 mM). After 3 days, [3H]-thymidine (1 mCi/well; Amersham Biosciences) was added, and the cells were cultured for an additional 16 hours. Then the cells were harvested onto fiberglass filters, and detected for their radioactivity in a Matrix-96 direct b counter (Packard Instrument Co.). Cell viability was determined by the MTS colorimetric assay using tetrazolium reagent. MCF-7 cells and MDA-MB 436 cells (56103/500 ml) were cultured in serum-free RPMI with indicated agents. After 4 days, 20 ml of MTS solution were added to each well and incubated at 37uC for 2 hours. Absorbance at 490 nm, which is directly proportional to the number of living cells, was measured on a 96-well plate reader. Data are expressed as a percentage of the untreated cells cultivated under the same conditions. Tumor xenografts Four groups of eight female BALB/c-nu mice each were introduced to the experiment. MDA-MB-436 (26106) breast cancer cells that were untransfected or transfected with GFP- shRNA or PKCf-shRNA were inoculated into the mammary fat pads of female BALB/c-nu mice. When the xenografts were palpable (approximately 5 mm in diameter), intratumoral injec- tion of PBS or 30 mg/kg HGF was performed biweekly for four consecutive weeks. Tumor growth was evaluated by monitoring the tumor volume (TV = length6width260.5) every three days for eight weeks. The animals were sacrificed when the xenografts reached 1.5 cm in diameter, and the mouse tumor xenografts, lungs and livers were harvested for further evaluation. Cryosec- tions (4 mm) of the harvested organs were stained with hematox- ylin and eosin (HE) for histological assessment, and total RNA was extracted for qRT-PCR analysis of human HPRT mRNA expression. Statistics All in vitro experiments were performed either in triplicate or in quintuplicate. Standard software 13.0 (SPSS, Chicago, IL) was used for statistical analyses. Measurement data were presented as mean 6 standard deviation (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA). The differences between the means were tested by an independent sample t-test or Bonferroni’s multiple comparison t-test. The x2 test was used to compare percentages. The significance level used was P,0.05. Supporting Information Figure S1 HGF upregulates CXCR4 expression and membrane presentation in human breast cancer cells. (A). Western blotting analysis revealed that the protein levels of CXCR4 and p-CXCR4 treated with HGF were higher than those treated with SDF in MDA-MB- 436 (left panel) and MCF-7 cells (right panel). Data are shown in arbitrary units (AU) normalized to PBS as the mean 6 SD of three independent experiments. # P,0.01 as compared to PBS. (B). Western blotting analysis revealed that the protein levels of Met and p-Met treated with HGF were higher than those treated with SDF in MDA-MB- 436 (left panel) and MCF-7 cells (right panel). Data are shown in arbitrary units (AU) normalized to PBS as the mean 6 SD of three independent experiments. # P,0.01 as compared to PBS. (C).Time course of relative CXCR4 content in two breast cancer cells following 50 ng/ml HGF stimulation. Data are shown in PKCf Induced by HGF Increases CXCR4 Expression arbitrary units (AU) normalized to PBS as the mean 6 SD of three independent experiments. # P,0.01 as compared to PBS. (TIF) Figure S2 HGF-induced increase in CXCR4 expression depends on PKCf activity. (A). Time course of relative p- PKCf levels as determined by immunoblot in MDA-MB-436 (left panel) and MCF-7 cells(right panel) following stimulation with 50 ng/ml HGF. Data are shown in arbitrary units (AU) normalized to PBS as the mean 6 SD of three independent experiments. # P,0.01, *P,0.05 as compared to PBS. (B).100 mM PKCf-siRNA1 (si1) or PKCf-siRNA2 (si2) transient transfections and siRNA-mediated PKCf protein silencing in MDA-MB-436 cells. Data are shown in arbitrary units (AU) normalized to PBS as the mean 6 SD of three independent experiments. # P,0.01 as compared to PBS. (C). Western blot analysis of PKCf(left panel) and CXCR4(right panel)expression levels in MDA-MB-436 cells cultured for 24 hours with 50 ng/ml HGF or/and 100 mM PKCf-siRNA1 (si1) or PKCf-siRNA2 (si2). Data are shown in arbitrary units (AU) normalized to PBS as the mean 6 SD of three independent experiments. # P,0.01 as compared to PBS. (D) and (E). Western blot analysis of total CXCR4 expression and p-CXCR4 levels in MDA-MB-436 cells cultured for 24 hours with 50 ng/ml HGF, 2 mM cholesterol sulfate (Choles sul), or PBS. As indicated, 10 mM PS of PKCf (PSf), PKCe (PSe), or PKCa/b (PSa/b) or 25 mM NSC23766 was used. Data are shown in arbitrary units (AU) normalized to PBS as the mean 6 SD of three independent experiments. # P,0.01*P,0.05 as compared to PBS. (F). Western blot analysis of Rac1-GTP and total-Rac1 in MDA-MB-436 and MCF-7 cells treated with 50 ng/ml HGF with or without 25 mM NSC23766. The experiment was repeated three times with similar results. A representative study is shown. # P,0.01 as compared to PBS. (G). Dose-dependent inhibition of HGF-induced p-PKCf was achieved using NSC23766 in HGF-treated MDA-MB-436 cells; the cells were assayed by Western blot. Data are shown in arbitrary units (AU) normalized to PBS as the mean 6 SD of three independent experiments. # P,0.01 as compared to PBS. (H). Western blot analysis of Rac1, PKCf and CXCR4 expression in MDA-MB-436 cells treated with 100 mM Rac1-siRNA for 48 hours. Data are shown in arbitrary units (AU) normalized to PBS as the mean 6 SD of three independent experiments. # indicates P,0.01 as compared to PBS. (TIF) Figure S3 Overexpressed CXCR4 in HGF-stimulated MDA-MB-436 cells is functional. (A–B). MDA-MB-436 cells were treated with indicated agents determined by Boyden chamber assays. Cells were counted in triplicate wells and in three identical experiments. Data are shown in invasion index normalized to PBS as the mean 6 SD of three independent experiments. # P,0.01 as compared to PBS. (C). Effect of HGF on breast cancer cell proliferation. MDA-MB 436 and MCF-7 cell lines were cultured for 3 days in serum-free medium with HGF (50 ng/ml) and/or other indicated agents, [3H]-thymidine (1 mCi/ well; Amersham Biosciences) was added for an additional 16 hours. Cells were harvested onto fiberglass filters, and radioactivity was detected in a Matrix-96 direct b counter (Packard Instrument Co.) Results shown are representative of three independent experiments. (D). For the MDA-MB 436 and MCF-7 cells were cultured in the presence of HGF and/or other indicated agents for 4 days. Cell viability was determined by MTS assay and reported as a percent of untreated cells. Results shown are representative of three independent experiments. (E). Western blot analysis of the total protein expression levels of MT1-MMP in PLoS ONE | www.plosone.org 14 January 2012 | Volume 7 | Issue 1 | e29124