CXCL4 Integrins Angiogenesis Figure 4. Immobilized CXCL4 or CXCL4/CTF promotes avb3-CHO cell spreading, focal adhesion and stress fiber formation. A. avb3- CHO and mock-transfected CHO cells were plated for 4 h on coverslips that had been coated with (25 mg/ml) CXCL4, (25 mg/ml) CXCL4/CTF, (25 mg/ ml) CXCL4/CTF-S, (100 mg/ml) fibrinogen and (100 mg/ml) poly-D-lysine. After washing, the cells are fixed. The degree of cell spreading is seen from the phase contrast micrographs (left side). Then the cells were permeabilized and stained to visualize focal adhesion (vinculin staining, green), and actin stress fibers (Rhodamin-Phalloidin staining, red) by confocal microscopy (right side). B. Effects of function-blocking anti-integrin antibodies and EDTA and RGD-, RGE-peptides on cell spreading to CXCL4 or CXCL4/CTF. avb3-CHO cells were incubated with 10 mM EDTA or 25 mg/ml of RGD- or RGE-peptides or 10 mg/ml anti-avb3 (LM609), before plating to coverslips coated with (25 mg/ml) CXCL4 or (25 mg/ml) CXCL4/CTF for 3 h at 37uC. Wells were washed with PBS, and cells were photographed. doi:10.1371/journal.pone.0002657.g004 PLoS ONE | www.plosone.org 6 July 2008 | Volume 3 | Issue 7 | e2657