Comparison of explant cultures of VSMCs from the aortic tissues of WT and OPN-KO mice.

<p>(A) and (B), Explant cultures of the aortic tissues of WT and OPN-KO mice for 3 days were performed with or without PDGF. Sprouting VSMCs were shown in the photographs. Microscope images are representative of 5 independent experiments. (C) and (D), Sprouting cells from the aortic tissues were counted and relative numbers to 0 day were expressed as the means ± SEMs of 5 independent experiments. **<i>P</i><0.01 vs. corresponding value at 0 day, ^##<i>P</i><0.05 vs. WT.</p

Role of OPN during PDGF-induced VSMC proliferation.

<p>(A) Cells were stimulated with the indicated concentrations of PDGF for 48 hrs, and then MTT assays were performed. Relative cell proliferation was expressed as the means ± SEMs of 8 independent experiments. *<i>P</i><0.05 and **<i>P</i><0.01 vs. control. (B) Cells were pre-treated with the indicated doses of MPIIIB10 (a neutralizing monoclonal antibody for OPN) or IgG for 1 hr, and cell proliferation was assessed using a MTT assay. Relative cell proliferation was expressed as the means ± SEMs of 4 independent experiments. **<i>P</i><0.01 vs. control, ^#<i>P</i><0.05 and ^##<i>P<</i>0.01 <i>vs</i>. vehicle.</p

Characteristics of OPN expression in PDGF-stimulated VSMCs.

<p>(A) VSMCs were stimulated with 10 ng/ml of PDGF for the indicated times, and OPN mRNA and protein levels were then assessed by RT-PCR and Western blotting. Images are representative of 4–6 independent experiments. MW, molecular weight marker (kDa). (B) Relative intensities of OPN mRNA and protein versus GAPDH and β-actin were quantified. Results were expressed as the means ± SEMs of 4–6 independent experiments. *<i>P</i><0.05 and **<i>P</i><0.01 vs. corresponding value at 0 hr.</p

Identification of ICB-targeted transcription factors in VSMCs stimulated with PDGF.

<p>(A) Cells were transiently tansfected with pLuc-OPN-538 constructs for 24 hrs, pretreated with the indicated doses of ICB, and then stimulated with PDGF (10 ng/ml) for 4 hrs. Relative luciferase activities were presented as the means ± SEM of 8 independent experiments. **<i>P</i><0.01 vs. control, ^#<i>P</i><0.05 and ^##<i>P</i><0.01 vs. vehicle. (B) The effects of ICB on the bindings of active AP-1 and C/EBPβ were assessed using a ChIP assay. IgG was used as negative control. Images are representative of 4 independent experiments. Quantitative results were expressed as the means ± SEMs of 4 independent experiments. **<i>P</i><0.01 vs. corresponding control, ^#<i>P</i><0.05 and ^##<i>P</i><0.01 vs. corresponding vehicle.</p

Effects of ICB on PDGF-induced VSMC proliferation.

<p>(A) Cells were pre-treated with the indicated doses of ICB (10 and 30 μg/ml) for 4 hrs and then stimulated with 10 ng/ml of PDGF for 48 hrs. Microscope images are representative of 8 independent experiments. (B) Cells in culture plate were counted and relative cell numbers to control were expressed as the means ± SEMs of 8 independent experiments. **<i>P</i><0.01 vs. control, ^#<i>P</i><0.05 and ^##<i>P<</i>0.01 <i>vs</i>. vehicle. (C) Cell proliferation was assessed using a MTT assay. Relative cell proliferation was expressed as the means ± SEMs of 8 independent experiments. **<i>P</i><0.01 vs. control, ^#<i>P</i><0.05 and ^##<i>P<</i>0.01 <i>vs</i>. vehicle.</p

Identification of transcription factors involved in PDGF-induced OPN expression in VSMCs.

<p>(A) Cells were transiently transfected with various promoter constructs or an empty luciferase vector (pGL3) for 24 hrs, and then stimulated with PDGF (10 ng/ml) for 4 hrs. Relative luciferase activities were presented as the means ± SEMs of 12 independent experiments. **<i>P</i><0.01 vs. non-treated control. (B) Nucleotide sequence of the -538 ~ -234 promoter region of the OPN gene. The transcription factor binding sites were identified using TFSearch software. The sequences of potential binding sites for AP-1 and C/EBPβ in pLuc-OPN-538 were underlined. (C) The bindings of AP-1 and C/EBPβ in PDGF-treated VSMCs were assessed using a ChIP assay. IgG was used as negative control. The images are representative of 4 independent experiments. Quantitative results were expressed as the means ± SEMs of 4 independent experiments. *<i>P</i><0.05 and **<i>P</i><0.01 vs. value at 0 hr.</p

Effects of ICB on PDGF-induced OPN expression in VSMCs.

<p>(A) Cells were pre-treated with the indicated doses of ICB for 4 hrs, and then stimulated with 10 ng/ml of PDGF for 12 hrs. OPN mRNA and protein levels were assessed by RT-PCR and Western blotting. Images are representative of 5–7 independent experiments. (B) Relative intensities of OPN mRNA and protein versus GAPDH and β-actin were expressed as the means ± SEMs of 5–7 independent experiments. **<i>P</i><0.01 vs. corresponding control, ^#<i>P</i><0.05 and ^##<i>P<</i>0.01 <i>vs</i>. corresponding vehicle.</p

Effects of 1-methoxyoctadecan-1-ol on infarct volume, neurological evaluation, and wire-grip test in a photothrombotic ischemic model.

<p>Mice received intraperitoneal administration of DMSO or 1, 3/kg of 1-methoxyoctadecan-1-ol at 30 min before the ischemic insult. Representative photographs of coronal brain sections stained with TTC in vehicle (Veh)- and 1-methoxyoctadecan-1-ol-treated mice (A). White indicates the infarct area. Quantification of the infarct volume, neurological score, and wire-grip test (B). *<i>P</i><0.05, **<i>P</i><0.01 <i>vs</i>. vehicle group. Data are expressed as mean±SEM of three separate experiments.</p

Time-course effects of 1-methoxyoctadecan-1-ol on calpain activation, STEP cleavage, and p38 MAPK phosphorylation after glutamate exposure.

<p>Cortical neurons were treated with glutamate (300 µM, 15 min) (A and C) and pretreatment with 1-methoxyoctadecan-1-ol (0.1 µg/ml, 24 h) followed by exposure to glutamate (B and D) and both were maintained in the original medium for the specified time. Equal amounts of proteins and each sample were subjected to Western blot assays using the indicated antibodies. Equal protein loading was confirmed by actin expression.</p

Flow cytometry analysis for neuronal death.

<p>Cortical neurons were pretreated with 1-methoxyoctadecan-1-ol (0.01 and 0.1 µg/ml) for 24 h, followed by exposure to 200 µM glutamate for 6 h. Cells were harvested and stained with Annexin V-FITC/PI, as described under methods and analyzed using flow cytometry. Annexin V⁺PI⁻ cells indicate early apoptotic cells, whereas Annexin V⁺PI⁺ cells are late apoptotic cells. The estimates (%) of gated cells in different compartments are given for each dot blot. Representative flow cytometry analysis scatter-grams of Annexin V/PI staining (A) and quantitative analysis of the histograms (B and C).^#<i>P</i><0.05 and ^##<i>P</i><0.01 <i>vs</i>. control; *<i>P</i><0.05 <i>vs</i>. treatment with glutamate alone. Data are represented as the mean±SEM of three independent experiments.</p