Figure 7

CNN3 knockdown by siRNA treatment of lens epithelial explants induces reorganization of actin stress fibers, and increases α-SMA and fibronectin in association with increased Yap nuclear translocation and decreased E-Cadherin. (A). Treatment of mouse lens epithelial explants with CNN3 siRNA for 72 hours induces changes in cell morphology from the epithelial hexagonal shape to an elongated morphology with reorganization of actin stress fibers (TRITC-phalloidin staining) distributing transversely throughout the cell body, reorganization of p-MLC similar to actin stress fibers, a robust increase in α-SMA and fibronectin levels and translocation of Yap into the nucleus and decrease in E-cadherin staining compared to cells treated with a scrambled siRNA as evidenced by immunofluorescence evaluation. Scale bars indicate image magnification. (B,C) Immunoblot based quantification under the above described conditions showed a significant decrease (*P < 0.05; n = 4; Student t test) in CNN3 and ~90% (n = 2) decrease in E-cadherin protein levels, and an increase in the levels of α-SMA and fibronectin (n = 4; *P < 0.05; Student t test) in CNN3 siRNA treated epithelial explants compared to the respective controls. GAPDH was immunoblotted as a loading control. Data for two representative samples (Lanes 1 and 2) are provided in panel B. Sc and Scr siRNA: Scrambled siRNA.