KLF2 is essential for shear fiber formation, cell alignment, and inhibition of JNK signaling in vitro, and these shear fibers are very similar in endothelium in vivo. (A) HUVECs transduced with either control siRNA lentivirus (filled bars) or KLF2-silencing lentivirus (open bars) were subjected to pulsatile shear stress and fixed. Differential interference contrast microscopy was performed, and approximately 100 cells per condition in 3 independent experiments were analyzed. Cell angle compared with the direction of the flow was measured and visualized in a bar graph. Differences in cell angles in the absence of KLF2 are not significant. (B) HUVECs were treated as in panel A, and immunofluorescence microscopy was performed as for Figure 5B. These images were acquired by merging the green channel (vinculin), the red channel (F-actin), which was acquired at 1 μm higher magnification than the green channel, and the blue channel (nuclei), which was taken at 4 μm higher magnification than the red channel. The flow direction was from left to right. The arrowheads indicate short basal actin filaments similar to those observed after KLF2 overexpression. (C) Intact rat renal arteries and saphenous veins were stained with phalloidin-TRITC (F-Actin, red) and Hoechst 33342 (Nuclei, blue) in situ and visualized by the use of immunofluorescence microscopy and computational deconvolution. The arrowheads show short basal actin filaments, similar to those observed after KLF2 overexpression. (D) Western blot analysis of P-JNK1 in HUVECs treated as in panels A and B. α-Tubulin was used as a loading control. Quantifications are shown for 4 independent experiments. *P < .05.