ILK prevents endothelial-to-mesenchymal transition (EndMT) in human valve endothelial cells (hVECs). (A) Immunohistochemistry of ILK in the aortic and ventricular sides of the endothelial layers of non-CAVD or CAVD human aortic valve leaflets. Central and right panels are magnifications of the areas enclosed in squares in the left panels. A = aortic side, V = ventricular side. Scale bar = 100 µm (left panel); 10 μm (central and right panels). Quantification of ILK expression is shown below. n = 6. *** p < 0.001 vs. non-CAVD; * p < 0.01 vs. V side CAVD. (B) Western blot and quantification of ILK protein expression in isolated non-CAVD or CAVD human valve endothelial cells (hVECs). Each point represents ILK expression in VECs isolated from different patients. n = 6. * p < 0.01 vs. non-CAVD. (C) Western blot analysis and quantification of ILK and the EndMT markers CD31, von Willebrand Factor (vWF), alpha-smooth muscle actin (α-SMA), transgelin (SM22α), and Snail in hVECs transfected with siRNA Scramble (si Sc) or siRNA ILK (si ILK) for 5 or 7 days. n = 11, CD31, αSMA, and ILK; n = 6, Snail, SM22α, and vWF. * p < 0.01 vs. Si Sc; ** p < 0.001 vs. Si Sc; *** p < 0.001 vs. Si Sc. (D) Confocal fluorescent microscopy images (left) and quantification of nuclear fluorescence intensity (Nuclear FI) (right) of Snail (green) in transfected hVECs for 5 days. Cell nuclei were counterstained with Hoechst. Scale bar = 10 µm. n = 6. * p < 0.01 vs. Si Sc.