MDA-MB-436 cells cultured for 24 hours with the indicated agents. 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. (TIF) Figure S4 HGF results in PI3K/Akt pathway phosphor- ylation and activates PKCf phosphorylation. (A). Detec- tion of phosphorylated Akt or of the respective total Akt protein expression or Rac1-GTP and total-Rac1 by western blot analysis. MDA-MB-436 cells were left 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. 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). 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. Data are shown in arbitrary units (AU) normalized to PBS as the mean 6 SD of three independent experiments. * indicates P,0.05, # indicates P,0.01 as compared to PBS. (TIF) Figure S5 Treatment with HGF slightly enhanced the tumor growth of breast cancer xenografts in BALB/c-nu mice. Tumor volume in the mammary fat pads was monitored in BALB/c-nu mice xenografted with MDA-MB-436 (26106 cells) breast cancer cells that were uninfected or infected with GFP- shRNA or PKCf-shRNA. Biweekly intratumoral injection with PBS or 30 mg/kg HGF was performed for 4 consecutive weeks once the xenografts were palpable (around 5 mm in diameter). The number of mice with detectable tumors is indicated. (TIF) Figure S6 HGF enhances CXCR4 expression via PKCf and promotes the invasion and metastasis of breast cancers in BALB/c-nu mice. (A–B) Immunoblot of total PKCf and p-PKCf (A) or total CXCR4 and p-CXCR4 (B) protein, respectively, in breast tumor xenografts in female nude mice that were inoculated in the mammary fat pads with MDA- References 1. Chen J, Yao Y, Gong C, Yu F, Su S, et al. CCL18 from tumor-associated macrophages promotes breast cancer metastasis via PITPNM3. Cancer Cell 19: 541–555. 2. Yu F, Yao H, Zhu P, Zhang X, Pan Q, et al. (2007) let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 131: 1109–1123. 3. Balkwill F (2004) The significance of cancer cell expression of the chemokine receptor CXCR4. Semin Cancer Biol 14: 171–179. 4. Balkwill F (2004) Cancer and the chemokine network. Nat Rev Cancer 4: 540–550. 5. Meier R, Muhlethaler-Mottet A, Flahaut M, Coulon A, Fusco C, et al. 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