Figure 4.

AsSn alters RhoA expression and activity, correlating with cell invasion. (A) Transient transfection of AsSn results show two to three-fold repression of RhoA promoter activity by Snail downregulation. (B) RhoGTPase levels in the lysates of mock-transfected and AsSn cells were analyzed by western blotting with β-actin serving as a quantitative control. The activities of the enzymes were determined by a pull-down assay using GST-C21 to detect Act-RhoA or with GST-PAK-CD for active Rac1 and Cdc42. Overexpression of AsSn led to a significant decrease of RhoA activity. (C) Mock-transfected and AsSn cells were transfected with expression vectors coding for the Wt, DN or Act forms of RhoA-GFP fusion proteins or GFP alone (GFP). Two days after transfection, cells were subjected to a Matrigel invasion assay. DN-RhoA significantly augmented the inhibitory effect of AsSn in cell invasion (*P<0.05). (D) Cells were lysed to assess the expression of fusion proteins by immunoblotting with anti-GFP or anti-RhoA antibody. (E) Mock-transfected and AsSn cells were transfected with expression vectors coding for Wt- or DN-RhoA-GFP fusion proteins. Two days after transfection, cells were lysed and subjected to immunoblotting with anti-MMP-2 antibody. MMP-2 expression and activity was decreased in cells transfected with DN-RhoA and increased in cells transfected with Act-RhoA. AsSn, antisense-Snail cDNA construct; Wt, wild-type; DN, dominant-negative; Act, active.