FIGURE 5.

Inhibition of CES1 impacts MDA-MB-231 cell morphology and RhoA activation status. A, inhibition of CES1 alters cell morphology of MDA-MB-231 cells. Cells were treated for 72 h with luciferase (LUC) or CES1 siRNA as described under “Experimental Procedures.” Cells were then plated on coverslips coated with fibronectin and allowed to adhere for 2 h prior to imaging. Representative images are displayed showing the more rounded morphology and altered actin cytoskeletal structure in cells treated with siRNA targeting CES1 as compared with those treated with siRNA targeting luciferase (LUC). B, knockdown of CES1 increases RhoA-GTP levels. MDA-MB-231 cells were treated with luciferase (LUC) or CES1 siRNA for 72 h as described above and then plated on dishes coated with fibronectin for 2 h. Lysates were prepared and activated RhoA (RhoA-GTP) was isolated by pulldown with GST-Rhotekin as described under “Experimental Procedures.” Levels of precipitated and total (Tot.) RhoA were determined by immunoblot analysis using a RhoA-specific antibody; β-actin was utilized as a loading control. C, quantitative representation of data in B. The level of activated RhoA is expressed as a fraction of total RhoA l. Data represent the mean ± S.E. from three independent experiments.