Figure 5

Downregulation of CNN3 expression induces changes in cell shape, reorganization of the actin cytoskeleton, increase focal adhesions and decreases in E-cadherin and β-catenin in lens epithelial cell cultures. (A) siRNA-mediated downregulation of CNN3 expression in serum starved mouse lens epithelial cells exhibit an altered, elongated and contractile morphology (phase contrast images) compared to control cells. (B,C) Downregulation of CNN3 expression in siRNA treated mouse lens epithelial cells was confirmed by a significant (*P < 0.01; Student t test) decrease in CNN3 protein levels relative to cells treated with scrambled control siRNA, as evidenced by immunoblotting analysis. (D) CNN3 deficient epithelial cells reveal reorganization of actin stress fibers, and increased focal adhesions formation based on increased immunofluorescence of phospho-paxillin and phospho-FAK. These cells also reveal a decrease in cell-cell adhesion based on the decrease in E-cadherin and β-catenin immunofluorescence relative to control cells. Additionally, CNN3 deficient lens epithelial cells reveal significant (*P < 0.01; Student t test) increases in the levels of p-paxillin, P-FAK and p-MYPT1 with a concomitant decrease in E-cadherin and β-catenin levels but no change in phospho-MLC relative to controls, based on immunoblotting analyses (Panel E) and densitometric quantification (Panel F). GAPDH was probed as a loading control. Sc and Scr siRNA: Scrambled siRNA. Scale bars indicate image magnification.