Clone 11g8

The in vitro invasion assay was performed using the Bio-Coat Matrigel invasion assay system (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. Hep3B, Huh7 and Hep3B cells (2×10⁵ cells) were suspended in serum-free medium containing 0.5% BSA and seeded into the Matrigel transwell chambers consisting of polycarbonate membranes with 8-µm pores. After pre-incubation with an anti-CXCR4 antibody (10 µg/ml, Clone 12G5; R&D Systems); an anti-CXCR7 antibody (10 µg/ml, Clone 11G8; R&D Systems); or a specific CXCR4 antagonist, Peptide R (5 µM), which was developed in our laboratory, for 45–60 minutes at 37 °C. Then, CXCL12 or CXCL11 (100 ng/ml) was added to the lower chamber. After 16 h at 37 °C, the cells on the upper surface of the filter were removed using a cotton wool swab. The cells were counted in ten different consecutive high-power fields (magnification: ×200). The anti-CXCR7 monoclonal antibody CXCR7/RDC-1 (Clone 11G8) and the anti-CXCR4 monoclonal antibody against human CXCR4 (Clone 12G5) were purchased from R&D Systems.

The migration of HepG2, Hep3B, Huh7, SNU398, SNU449 and SNU475 cells was assayed in 24-well transwell chambers (Corning Inc., Corning, NY, USA) using inserts with an 8-µm pore membrane. The membranes were pre-coated with collagen (human collagen type IV) and fibronectin (10 µg/ml). Cells were placed in the upper chamber (2.0×10⁵ cells/well) in assay medium (Dulbecco's modified Eagle medium) containing 0.5% BSA and were incubated with a CXCR4 inhibitor, AMD3100 (5 µM; Sigma, St Louis, MO, USA); with a CXCR7 inhibitor, CCX771 (500 nM, gift from ChemoCentryx, Mountain View, CA, USA); with a specific CXCR4 antagonist, Peptide R (5 µM), developed in our laboratory; with an anti-CXCR7 monoclonal antibody, CXCR7/RDC-1 (10 µg/ml, Clone 11G8); or with an anti-CXCR4 monoclonal antibody (10 µg/ml, Clone 12G5; R&D Systems) for 45–60 min at 37 °C. Then, CXCL12 or CXCL11 (100 ng/ml) was added to the lower chamber. After 16 h at 37 °C, the cells on the upper surface of the filter were removed using a cotton wool swab. The cells were counted in ten different consecutive high-power fields (magnification: ×200).

Cells grown on coverslips (Paul Marienfeld Gmbh & Co. KG, Lauda-Koenigshofen, Germany) were washed with cold phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde (Sigma-Aldrich Co., Saint Louis, MO, USA) for 15 min at 37℃, and washed 3 times with PBS. The cells were then incubated with a murine monoclonal anti-CXCR4 antibody (1:2,000; clone 12G5; R&D Systems, Minneapolis, MN, USA) and a murine monoclonal anti-CXCR7/RDC-1 antibody (1:2,000; clone 11G8; R&D Systems, Minneapolis, MN, USA) for 90 min at 37℃, washed 3 times with PBS, and incubated with FITC- or PE-conjugated anti-mouse IgG (1:4,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) at 37℃ for 60 min. The cells were washed 3 times with PBS, fixed, mounted on glass slides with PBS, and observed under a laser scanning confocal microscope (Olympus Corp., Lake Success, NY, USA).