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. 2010 Oct 5;285(50):38832–38840. doi: 10.1074/jbc.M110.124958

FIGURE 3.

FIGURE 3.

Effects of Cav-1 overexpression and knockdown on cell migration and invasion. H460 cells were stably transfected with Cav-1 or control plasmid as described under “Materials and Methods.” A, Cav-1 expression in the control and Cav-1-transfected cells was determined by Western blotting. Cell extracts were prepared and separated on 10% SDS-polyacrylamide gels, transferred, and probed with Cav-1 antibody. β-Actin was used as a loading control. B, effect of Cav-1 overexpression on cell migration. Cav-1 and control-transfected cells were cultured in 24-well plates and analyzed for cell migration at 24 h by wound assay. C, effect of Cav-1 overexpression on cell invasion. Cav-1 and control-transfected cells were added to extracellular matrix-coated inserts in a Transwell chamber and incubated for 24 h. Invading cells were counted under a fluorescence microscope after staining with Hoechst 33342, and the average number of cells was scored in each case. D–F, Cav-1 knockdown experiments were performed using H460 cells treated with Cav-1 shRNA (shCav-1) viral particles or control shRNA (shCon) particles as described under “Materials and Methods.” D, Cav-1 expression in shCav-1 and shCon-treated cells determined by Western blotting at 36 h post-treatment (left panel). Rescue experiment was performed in shCav-1-treated cells by transfecting the cells with Cav-1 plasmid as described above and analyzed for Cav-1 expression by Western blotting (right panel). E and F, migration and invasion of shCon, shCav-1, and rescued cells determined by wound and Transwell assays, respectively. Data are the mean ± S.D. (n = 3). *, p < 0.05 versus control transfection; #, p < 0.05 versus shCav1 control.