Effects of p66^Shc silencing on endothelial activation in HUVEC.

<p><i>A</i>. (top left) Representative immunoblot of p66^Shc Ser³⁶ phosphorylation in HUVEC transfected with 50 nM or 100 nM of siRNA#1 or siRNA#2, and (right) quantification of multiple experiments. <i>Hatched </i><i>bar</i>, cells transfected with a negative control siRNA; black bars, cells treated with siRNA#1 and siRNA#2. (bottom left) Representative immunoblot of p66^Shc protein content in HUVEC transfected with 50 nM or 100 nM of siRNA#1 or siRNA#2, respectively, and (right) quantification of multiple experiments. <i>Hatched </i><i>bar</i>, cells transfected with a negative control siRNA; grey bars, cells treated with siRNA#1; light grey bars, cells treated with siRNA#2. *<i>P</i><0.05 vs. cells transfected with negative siRNA. Neg., negative siRNA. <i>B</i>. Leukocyte transmigration test. HL-60 cells were added to control HUVEC (w.t., Lipofectamine™, and negative siRNA) and HUVEC treated with p66^Shc siRNA#1 (100 nM) or p66^Shc siRNA#2 (50 nM), and left to migrate for 18 h at 37°C. Studies were carried out under basal conditions or following stimulation with 50 ng/ml TNFα for 4 h. Migrated cells were stained and measured by OD at 450 nm. Data represents the mean of triplicates from two independent experiments, and are normalized to basal HUVEC/wt. *<i>P</i><0.05 vs. no TNFα; ^§P<0.05 vs. HUVEC/wt. <i>C</i>. Effects of siRNA-mediated knockdown of p66^Shc on E-Selectin mRNA levels under basal conditions (left) and following TNFα stimulation (right). HUVEC were transfected with 100 nM siRNA#1 (grey bars) or 50 nM siRNA#2 (light grey bars), and then left untreated or incubated with 50 ng/ml TNFα for 1 h. *<i>P</i><0.05 vs. basal; ^#<i>P</i><0.05 vs. TNFα-stimulated controls (wild-type, Lipofectamine™, and negative siRNA). Lysates were analyzed by qPCR 48 h following transfection, using β-actin as internal control. Lipo, Lipofectamine™.