Effects of AST and ASIV on TNFα-induced up-regulation of ICAM-1 and E-selectin mRNAs.

<p>Confluent mAECs were pre-incubated with 250 µg/mL of AST or ASIV for 2 h, then washed and incubated for 6 h with 30 ng/mL TNFα. Cells were then collected and analyzed for ICAM-1 expression using real-time qPCR. Data are representative of three independent experiments with determinations made in triplicate and shown as mean values+S.D. **<i>P</i><0.01 compared with cells treated with TNFα alone. CN: Control.</p

Effects of AST and ASIV on TNF-induced NF-κB-p65 phosphorylation and IκBα degradation.

<p>Confluent mAECs were pre-incubated with 250 µg/mL of AST or ASIV for 2 h, then washed and incubated for 30 min with 30 ng/mL TNFα. Cells were then collected and analyzed for expression of phosphorylated NF-kB-p65 and IκBα by Western blott analysis. Data are representative of three independent experiments with determinations made in triplicate and shown as mean values+S.D. **P<0.01 compared with cells treated with TNFα alone. CN: Control.</p

AST, but not ASIV, reduced surface expression of TNFR1 protein.

<p>Confluent mAECs were incubated with 250 µg/mL AST or ASIV for 2 h, then cell surface proteins were isolated and TNFR1 expressed on the cell surface was measured by Western blot analysis. Data were representative of three independent experiments with determinations made in triplicate and shown as mean values+S.D. **P<0.01 compared with cells treated with control group. CN: control.</p

Mouse arterial endothelial cells (mAECs).

<p>Cells were observed under light microscope (Left), or after fluorescence staining with vWF (Right). Cultured cells were fixed and treated with rabbit anti-mouse vWF antibody followed by FITC-conjugated anti-rabbit immunoglobulin G. vWF antigen was strongly identified in the cytoplasm (green). The cell nuclei were stained by Hoescht 33258 (blue). Scale bar: 20 µm.</p

Effects of AST and ASIV on TNF-induced NF-κB-p65 translocation.

<p>Confluent mAECs were pre-incubated with 250 µg/mL of AST or ASIV for 2 h, then washed and incubated for 50 min with 30 ng/mL of TNFα. The cells were then fixed and stained for NF-κB-p65 signal. Scale bar: 20 µm.</p

Effects of AST and ASIV on TNF-induced apoptosis.

<p>Confluent mAECs were pre-incubated with 250 µg/mL AST or ASIV for 2 h, then washed and incubated for 8 h with 30 ng/mL TNFα+10 µg/mL CHX (T/C). The cells were then photographed, collected and analyzed for the expression of cleaved-caspase 3 by Western blot analysis. Data were representative of three independent experiments with determinations made in triplicate and shown as mean values+S.D. * P<0.05, **P<0.01 compared with cells treated with TNF+CHX. CN: control; T/C: TNF+CHX.</p

Effects of ASI, ASII and ASIII on TNF-induced ICAM-1 expression and IκBα degradation.

<p>Confluent mAECs were pre-incubated with 250 µg/mL ASI, ASII or ASIII for 2 h, then washed and incubated for 6 h or 30 min with 30 ng/mL TNFα. Cells were then collected and analyzed for the expression of ICAM-1 mRNA or IκBα by performing real-time qPCR and Western blot analysis, respectively. Data were representative of three independent experiments with determinations made in triplicate and shown as mean values+S.D. * P<0.05, **P<0.01 compared with cells treated with TNFα. CN: Control.</p