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Fig. 6.
STAT3 transcriptionally activates ICAM-1, a cell adhesion molecule that promotes vascular permeability. (A) Top: the pGL3-ICAM1-WT plasmid containing the human ICAM-1 promoter with a STAT3 binding site located at −115 to −107 bp. Bottom: the pGL3-ICAM1-SDM plasmid with a site-directed mutation (SDM) in the STAT3 binding site as indicated. (B) Dual luciferase assays were performed in HUVECs that were transfected with pGL3-ICAM1-WT or pGL3-ICAM1-SDM and empty vector or constitutively active STAT3. Firefly and Renilla luminescence was measured and plotted as a ratio. Mean±s.e.m., one-way ANOVA followed by Bonferroni test. n=9 technical replicates. Depicted findings are representative of three independent experiments. (C) HUVECs that had been stably transduced with lentivirus encoding STAT3-specific shRNA or control shRNA were stimulated with human VEGF-165 protein (25 ng/ml) and the lysates were immunoblotted for ICAM1, p-STAT3 (Y705) and total STAT3. Depicted data are representative of three biological replicates. (D) RNA was harvested from VEGF; Stat3+/+ or VEGF; Stat3−/− 3 dpf embryos for quantitative PCR. stat3 transcripts are reduced in VEGF; Stat3−/− (n=5) compared to VEGF; Stat3+/+ zebrafish (n=7). Mean±s.e.m., unpaired, two-tailed Student's t-test. (E) The expression of icam-1 was assessed by real-time quantitative PCR using RNA derived from each zebrafish embryo in the absence of VEGF induction (Stat3+/+, n=3; Stat3−/−, n=2) or 8 h following VEGF induction (Stat3+/+, n=4; Stat3−/−, n=3) in the heat-inducible VEGF; Stat3 mutant zebrafish. Mean±s.e.m., one-way ANOVA followed by Bonferroni test.