Fig. 5. Effects of echinochrome A (Ech A) on OPN expression in HMGB1-stimulated VSMCs.
(A) The effects of Ech A on the binding activity of AP-1 on the OPN promoter were assessed using a ChIP assay. IgG was used as the negative control. The relative binding intensity to input signals was quantified, and expressed as the means ± SEMs of 6 independent experiments. **p < 0.01 vs. non-treated control, #p < 0.05 and ##p < 0.01 vs. vehicle in the HMGB1-treated group. (B) Cells were transiently transfected with pLuc-OPN-538 constructs for 24 h, and then stimulated with HMGB1 (100 ng/ml) for 1 h in the presence of the indicated concentrations of Ech A (3 or 10 μM). The relative luciferase activities were presented as the means ± SEM of 6 independent experiments. **p < 0.01 vs. control, #p < 0.05 and ##p < 0.01 vs. vehicle in the HMGB1-treated group. (C) VSMCs were pretreated with Ech A (3 or 10 μM) for 24 h and then stimulated with HMGB1 (100 ng/ml) for 6 h and 48 h to determine mRNA and protein expression, respectively. GAPDH and β-actin were used as the internal controls for mRNA and protein expression, respectively. The relative band intensities were quantified, and expressed as the mean ± SEM of 6 independent experiments. **p < 0.01 vs. corresponding control, #p < 0.05 and ##p < 0.01 vs. corresponding value in vehicle of the HMGB1-treated group. OPN, osteopontin; VSMCs, vascular smooth muscle cells; HMGB1, high-mobility group box 1; AP-1, activator protein 1; ChIP, chromatin immunoprecipitation.
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