Fig. 1. Silencing Pfn1 expression affects the actin cytoskeleton in HUVECs: (A) Target specificity of Pfn1-siRNA was initially demonstrated by transfecting either control (C) or Pfn1-specific (P) siRNAs in MDA-MB-231 breast cancer cells that stably express either GFP or different point-mutants of GFP-Pfn1 (designated as mutant-1 and mutant-2 that carried a 2-base-pair alteration both within and outside of the targeting region of Pfn1-siRNA, respectively). Fluorescence micrographs of cells show that Pfn1-siRNA suppresses the expression of mutant-2, but not of mutant-1. Fluorescence of control GFP-expressing cells is not affected by Pfn1-siRNA treatment. Bar, 150 µm. (B) A bar graph shows time-dependent progressive loss of Pfn1 expression with nearly 97% suppression of Pfn1 expression 96 hours after Pfn1-siRNA transfection (data summarized based on immunoblot analyses of HUVEC extracts from three independent experiments). The inset shows the actual representative Pfn1-immunoblots of HUVEC extracts prepared at different time-points after siRNA transfection with GAPDH blot serving as the loading control. Additional immunoblot data shows no detectable Pfn2 expression under either experimental condition (purified Pfn2 serves the positive control for the immunoblot). (C) Rhodamine-phalloidin staining of HUVECs shows that silencing Pfn1 dramatically inhibits the formation of actin stress-fibers. Bar, 20 µm. (D) A bar graph displaying the relative (normalized with respect to the control cells) fluorescence intensity of phalloidin shows a 29% decrease in the average level of F-actin in Pfn1-deficient cells. These data were obtained from analyses of 640 control and 584 Pfn1-deficient cells from two independent experiments, the difference of which was found to be statistically significant (the asterisk indicates P<0.0001). (E) Immunoblots show comparable expression levels of actin and several ABPs such as VASP, mDia1, and N-WASP at 48, 72 and 96 hours after transfection.