PKCζ depletion by siRNA and DN-PKCζ inhibits d-flow and ONOO⁻-induced apoptosis. (A) HUVECs were transfected with control or PKCζ siRNA for 48 h and then stimulated with d-flow for 36 h followed by TUNEL staining. Images were recorded as described in Materials and methods after counterstaining with DAPI to visualize nuclei (bottom). Apoptotic nuclei appear green (top). Bars, 25 µm. (right) Quantification of apoptosis is shown as the percentage of TUNEL-positive cells. (B) HUVECs were transfected with control or PKCζ siRNA for 48 h (left) or transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h (center). Cells were then treated with 100 µM ONOO⁻ or vehicle for 8 h and assayed by TUNEL staining. (right) DN-PKCζ and reduced PKCζ expression were confirmed by Western blotting with anti-PKCζ. Data are from three separate experiments using two or more different EC preparations (**, P < 0.01). (C) After transduction of Ad-DN-PKCζ or Ad-LacZ for 24 h, HUVECs were treated with 100 µM ONOO⁻ for 9 and 18 h, and Western blotting with anti–cleaved caspase 3 was performed. DN-PKCζ expression and protein loading were assessed by Western blotting with anti-PKCζ (middle) and antitubulin (bottom). (right) Quantification of cleaved caspase 3 is expressed as the relative ratio compared with tubulin expression. Results are expressed as the relative percentage of untreated cells in the LacZ control (100%). n = 3. **, P < 0.01 compared with each control. Molecular masses are given in kilodaltons. Error bars are means ± SD.