Effects of TNFα on endothelial activation in HUVEC.

<p>Cells were incubated with 50 ng/ml TNFα for the indicated times or left untreated. <i>A</i>. Leukocyte transmigration test. HL-60 cells were added to HUVEC/wt, stimulated with 50 ng/ml TNFα for 4 h, and left to migrate for 18 h at 37°C. 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 control. *<i>P</i><0.05 vs. no TNFα. <i>B</i>. TNFα regulation of E-Selectin mRNA levels. E-Selectin mRNA levels were evaluated by qRT-PCR, and normalized using β-actin as internal control. <i>C</i>. TNFα regulation of E-Selectin protein levels. E-Selectin protein levels were evaluated by immunoblotting, using GAPDH as internal control. <i>D</i>. Leukocyte transmigration test. HL-60 cells were added to control HUVEC (treated with negative siRNA) and HUVEC treated with E-Selectin siRNA#1 (50 nM) or siRNA#2 (100 nM) for 48 h, and left to migrate for 18 h at 37°C. Studies were carried out under basal conditions. Migrated cells were stained and measured by OD at 450 nm. Data represents the mean of triplicates of one experiment and are normalized to control. *<i>P</i><0.05 vs. negative siRNA. <i>E</i>. Effects of SP600125 (left) and PD98059 (right) on TNFα-induced E-Selectin mRNA expression. Cells were pre-treated with 30 mM JNK or MEK inhibitor, respectively, for 2 h and then exposed 50 ng/ml TNFα for the indicated times (untreated cells, black; inhibitor-treated cells, grey; DMSO-treated cells, light grey). E-Selectin mRNA levels were evaluated by qRT-PCR, using β-actin as internal control. *<i>P</i><0.05 vs. basal; ^#<i>P</i><0.05 vs. TNFα-stimulated cells.</p

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™.</p

Role of Ser³⁶ phosphorylation of p66^Shc in endothelial activation.

<p><i>A</i>. Basal and TNFα-stimulated phosphorylation of p66^Shc on Ser³⁶ in HUVEC overexpressing the p66^Shc Ala³⁶ mutant (HUVEC/p66^ShcAla³⁶). Cells were stimulated with 50 ng/ml TNFα for 0.5 h or left untreated. When indicated, cells were pre-incubated with 30 mM SP600125 or DMSO for 2 h prior to TNFα stimulation. Each pair of representative immunoblots shows p66^Shc phosphorylation on Ser³⁶ (top left) and Shc protein content (bottom left) in HUVEC/wt, HUVEC/p66^Shc, HUVEC/p66^ShcAla³⁶, and HUVEC/mock, respectively. The ratio of phosphorylated to total p66^Shc protein in the four cell lines is also shown (<i>right</i>; HUVEC/wt, filled bars; HUVEC/p66^Shc, open bars; HUVEC/p66^ShcAla³⁶, grey bars; HUVEC/mock, light grey bars). *<i>P</i><0.05, TNFα-stimulated HUVEC/p66^Shc vs. HUVEC/p66^ShcAla³⁶; ^#<i>P</i><0.05, SP600125-treated HUVEC/p66^Shc vs. untreated HUVEC/p66^Shc. <i>B</i>. Leukocyte transmigration test. HL-60 cells were added to HUVEC/w.t. and HUVEC treated with Ad/p66^Shc, Ad/p66^ShcAla³⁶, or Ad/mock for 48 h, 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>P</i><0.05 vs. HUVEC/p66^ShcAla³⁶. <i>C</i>. Effects of Ser/Ala³⁶ mutation on E-Selectin mRNA levels in HUVEC. HUVEC/wt (filled bars), HUVEC/p66^Shc (open bars), HUVEC/<i>Ala</i>³⁶ (grey bars), and HUVEC/mock (light grey bars) were treated with 50 ng/ml TNFα for 1 h or left untreated. E-Selectin gene expression was evaluated using qRT-PCR. *<i>P</i><0.05, TNFα-stimulated cells vs. unstimulated cells; ^#<i>P</i><0.05, HUVEC/p66^Shc vs. controls. <i>D</i>. Cartoon illustrating the signaling pathway mediating the stimulatory effect of TNFα on E-Selectin gene expression in HUVEC. MKKs, MKK-7 and MKK-4; IKK, IκBα kinase; ROS, reactive oxygen species.</p

TNFα-induced phosphorylation of p66^Shc on Ser³⁶ in HUVEC.

<p><i>A</i>. Dose-response studies. Cells were incubated with TNFα for 0.5 h at the indicated doses or left untreated. Representative immunoblots of p66^Shc phosphorylation on Ser³⁶ (top left) and Shc protein content (bottom left), and ratio of phosphorylated to total p66^Shc protein (right). Cell lysates were analyzed by immunoblotting with specific antibodies. <i>B</i>. Time-course studies. Cells were incubated with 50 ng/ml TNFα for the indicated times or left untreated. Representative immunoblots of p66^Shc phosphorylation on Ser³⁶ (top left) and Shc protein content (bottom left), and ratio of phosphorylated to total p66^Shc protein (right). <i>C</i>. Effects of the JNK inhibitor SP600125 on TNFα-induced phosphorylation of p66^Shc on Ser³⁶. Cells were pre-treated with 30 mM SP600125 for 2 h and then exposed to 50 ng/ml TNFα for 0.5 h. Representative immunoblots of p66^Shc phosphorylation on Ser³⁶ (top left) and Shc protein content (middle left), and ratio of phosphorylated to total p66^Shc protein (<i>right</i>; untreated cells, black bars; inhibitor-treated cells, grey bars; DMSO-treated cells, white bars). <i>D</i>. Effects of the MEK inhibitor PD98059 on TNFα-induced phosphorylation of p66^Shc on Ser³⁶. Cells were pre-treated with 30 mM PD98059 for 2 h and then exposed to 50 ng/ml TNFα for 0.5 h. Representative immunoblots of p66^Shc phosphorylation on Ser³⁶ (top left) and Shc protein content (middle left), and ratio of phosphorylated to total p66^Shc protein (<i>right</i>; untreated cells, black bars; inhibitor-treated cells, grey bars; DMSO-treated cells, white bars). GAPDH protein content was used as loading control. *<i>P</i><0.05 vs. basal; ^#<i>P</i><0.05 vs. controls.</p

Role of oxidative stress in p66^Shc signaling.

<p><i>A</i>. Levels of reactive oxygen species (ROS) in HUVEC/p66^Shc and control cells (HUVEC/wt and HUVEC/mock). Cells were pre-incubated with or without 30 mM SP600125 for 2 h and then treated with 50 ng/ml TNFα for 0.5 h or left untreated; ROS levels were evaluated by fluorimetry (HUVEC/wt, filled bars; HUVEC/mock, grey bars; HUVEC/p66^Shc, open bars). *<i>P</i><0.05 vs. untreated cells; ^#<i>P</i><0.05 vs. cells stimulated with TNFα alone; all values of HUVEC/p66^Shc were significantly different (<i>P</i><0.05) vs. HUVEC/wt. Data from multiple independent experiments (n=6) are expressed as mean±SE. <i>B</i>. Effects of N-acetyl-cysteine (NAC) on TNFα-induced phosphorylation of p66^Shc on Ser³⁶. <i>C</i>. Effects of NAC on TNFα-induced E-Selectin expression. In <i>B</i> and <i>C</i>, HUVEC/wt (black bars), HUVEC/mock (grey bars), and HUVEC/p66^Shc (open bars) were preincubated with 10 mM NAC for 0.5 h before stimulation with 50 ng/ml TNFα. *<i>P</i><0.05 vs. untreated cells; ^#<i>P</i><0.05 vs. TNFα-stimulated cells not treated with NAC. <i>D</i>. Cells were coincubated with TNFα (50 ng/ml) and NADPH oxidase (Nox)-inhibitor 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2810, 5 µM) or rotenone (Rot, 10 µM) + thenoyltrifluoroacetone (TTFA, 10 µM) for 0.5 h. Representative immunoblots of p66^Shc phosphorylation on Ser³⁶ and GAPDH (left), and ratio of phosphorylated p66^Shc protein to GAPDH (<i>right</i>; untreated cells, black bars; TNFα-treated cells, grey bars). *<i>P</i><0.05 vs. basal; ^#<i>P</i><0.05 vs. TNFα-stimulated cells.</p

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

<p>A. Representative images of HUVEC/p66^Shc and HUVEC/mock. Cells were infected with the adenoviral constructs and analyzed by fluorescent microscopy. The green staining identifies cells expressing the green fluorescent protein (GFP) encoded by the recombinant adenovirus. Cellular morphology was evaluated by optical microscopy. B. Leukocyte transmigration test. HL-60 cells were added to HUVEC/wt and HUVEC transduced with Ad/p66^Shc or Ad/mock for 48 h, 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. *P<0.05 vs. no TNFα; ^§P<0.05 vs. controls. C. Effects of p66^Shc overexpression on E-Selectin mRNA levels in HUVEC. HUVEC/wt (filled bars), HUVEC/p66^Shc (open bars), and HUVEC/mock (grey bars) were treated with 50 ng/ml TNFα for 1 h or left untreated. When indicated, cells were pre-treated with 30 mM SP600125 or DMSO for 2 h. E-Selectin gene expression was evaluated using qRT-PCR. The inset (right) shows E-Selectin mRNA levels under basal conditions. *P<0.05, TNFα-stimulated cells vs. unstimulated cells; ^#P<0.05, HUVEC/p66^Shc vs. controls; ^§P<0.05, SP600125-treated cells vs. controls.</p