FIGURE 3.

GMFG overexpression enhances chemoattractant-stimulated cell migration and adhesion to FN. Human monocytes or THP-1 cells were transfected with GFP vector or GFP-tagged GMFG plasmid for 48 h. A, Western blotting analysis of expression of GMFG or GMFG-GFP in monocytes or THP-1 cells. α-Tubulin was used as a loading control. B, Transwell migration assays were performed on 5-μm pore filters coated with 10 μg/ml FN in transfected primary human monocytes or THP-1 cells in the absence (Ctrl) or presence of fMLP (100 nm) or SDF-1α (100 ng/ml). The number of migrated cells was quantitated after 3 h, as described under “Experimental Procedures.” Data represent the mean ± S.D. (error bars). *, p < 0.05 compared with control GFP-transfected cells. C, transfected human monocytes or THP-1 cells were subjected to adhesion assays on 10 μg/ml FN-coated wells (in triplicate samples) in the absence or presence of fMLP (100 nm) or SDF-1α (100 ng/ml). The number of attached cells was quantitated after 15 min, as described under “Experimental Procedures.” Data represent the mean ± S.D. *, p < 0.05 compared with control GFP-transfected cells. D, representative flow cytometry results for cell surface expression of α5- or β1-integrins in GFP- or GMFG-GFP-transfected human monocytes. Monocytes were incubated with antibodies to α5-integrin or β1-integrin (both the active and inactive forms, MEM101A) for 1 h at 4 °C. After washing, the cells were labeled with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody and subjected to flow cytometry analysis. E, Western blotting analysis of α5-integrin or β1-integrin in whole-cell lysates from GFP- or GMFG-GFP-transfected monocytes or THP-1 cells. α-Tubulin was used as a loading control.