FIGURE 7.

Par3 knockdown reverses the effect of Gα₁₃ siRNA on cell-cell adhesion and proteolytic invasion. A, CD18-tet-MT cells were plated on glass coverslips, transfected with siRNA against DDR1, Gα₁₃, or Par3, and grown in the presence of doxycycline for 48 h. Cells on coverslips were fixed, permeabilized, and stained for E-cadherin and Par3. B, CD18-tet-MT cells transfected with siRNA against Par3, Gα₁₃, or DDR1 were grown in three-dimensional collagen for 48 h. Collagenase-extracted cell lysates were then analyzed for E-cadherin, Par3, Gα₁₃, and DDR1 expression by Western blotting. C, CD-18-tet-MT cells were transfected with siRNA against Gα₁₃, Par3, or both Gα₁₃ and Par3. The cells were plated in suspension in a hanging drop for 18 h in the presence of doxycycline. Quantitation of the area of cell clusters was performed using ImageJ. D, CD18-tet-MT cells transfected with siRNA against Gα₁₃ and/or Par3 were grown in three-dimensional (3D) collagen in the presence of doxycycline and EGF (20 ng/ml) and allowed to invade for 72 h. Quantitation of invasion was analyzed as detailed in Fig. 2. The number of cell clusters demonstrating a half moon shape was also quantified. The statistics of the invasion assay and the number of half moon cell clusters were performed using one-way ANOVA followed by Dunnett's post-test. The statistics of the hanging drop assay were performed using Student's t test. ns, not significant; **, p < 0.01; ***, p < 0.001. The results are representative of three independent experiments.