KLF2 induces distinct actin filament formation in a Rho-kinase–independent manner. (A) Mock-transduced (left) and KLF2-transduced (right) HUVECs were fixed, and immunofluorescence microscopy was performed. Visualized in blue are nuclei (stained with Hoechst 33342), in red F-actin (phalloidin-tetramethyl rhodamine iso-thiocyanate [TRITC]), and in green focal adhesions (vinculin stained with Alexa647). The top images were taken at high-power magnification focused on the basal side of the cell, whereas the middle images were taken at approximately 4 μm higher magnification. The bottom images were also taken with focus on the basal side of the cell. Arrowheads indicate typical actin fibers for the given condition. (B) Mock- or KLF2-transduced HUVECs were stimulated for 24 hours with TNF-α (10 ng/mL, top left), Thrombin (1 U/mL, middle left), Y27632 (10μM, top right), or left unstimulated (bottom). Then, cells were fixed, and immunofluorescence microscopy was performed. Visualized in green are focal adhesions (vinculin), in red actin filaments (phalloidin-TRITC), and in blue nuclei (stained with Hoechst 33342; top 4 images) or P-MLC (bottom 2 images). (C) Mock- (left) and KLF2-transduced (right) HMVECs and HAECs were fixed, and immunofluorescence microscopy was performed. Visualized in blue are nuclei (stained with Hoechst 33342), in red F-actin (phalloidin-TRITC), and in green focal adhesions (vinculin stained with Alexa647).