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. 2016 Feb 19;291(16):8549–8564. doi: 10.1074/jbc.M115.674200

FIGURE 1.

FIGURE 1.

GMFG knockdown reduces chemotaxis in human monocytes. A, knockdown efficiency of GMFG siRNA in monocytes or THP-1 cells. Cells were transfected with a control negative siRNA (Ctrl) or GMFG siRNA, and lysates were examined by Western blotting. β-Actin was used as a loading control. B and C, Transwell migration assays were performed on 5-μm pore filters coated with 10 μg/ml FN in primary human monocytes (B) or THP-1 cells (C) transfected with control negative siRNA (Ctrl siRNA) or GMFG siRNA in response to vehicle alone (0.1% BSA) or increasing concentrations of fMLP (1–100 nm) or SDF-1α (1–100 ng/ml). The number of migrated cells was quantitated after 3 h, as described under “Experimental Procedures.” D and E, Transwell migration assays were performed in human monocytes or THP-1 cells cotransfected with control negative siRNA (Ctrl siRNA) or GMFG siRNA and GFP vector or GFP-GMFG vector in response to vehicle alone (0.1% BSA) or fMLP (100 nm) or SDF-1α (100 ng/ml). The number of migrated cells was quantitated after 3 h, as described under “Experimental Procedures.” F–I, EZ-TAXIScan chemotaxis toward fMLP (100 nm) or SDF-1α (100 ng/ml) in human monocytes or THP-1 cells transfected with control negative siRNA or GMFG siRNA. F and G, representative images of migrating cells in a gradient of fMLP or SDF-1α after 3 h. Scale bars, 20 μm. Data are representative of three experiments. H and I, migration speed and chemotactic index were quantified from the captured images during the course of the EZ-TAXIScan chemotaxis assay, as described under “Experimental Procedures.” J, cell surface expression of fMLP receptor (fMLP-R) or chemokine receptor CXCR4 in control negative siRNA- or GMFG siRNA-transfected human monocytes or THP-1 cells. Cells were stained with polyclonal anti-fMLP receptor or anti-CXCR4 antibodies for 1 h at 4 °C. After washing, the cells were labeled with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody and subjected to flow cytometry analysis. K, Western blotting analysis of GMFG, fMLP receptor, and chemokine receptor CXCR4 expression in whole-cell lysates from monocytes or THP-1 cells transfected with control negative siRNA or GMFG siRNA. β-Actin was used as a loading control. Data represent the mean ± S.D. (error bars) from three independent experiments. *, p < 0.05 compared with control siRNA-transfected cells. **, p < 0.01 compared with control siRNA-transfected cells.