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[Preprint]. 2020 Nov 20:2020.10.27.358374. Originally published 2020 Oct 28. [Version 3] doi: 10.1101/2020.10.27.358374

Figure 7: STAT3 transcriptionally activates ICAM1, a cell adhesion molecule that promotes vascular permeability.

Figure 7:

(A) The pGL3-ICAM1-WT plasmid (top) containing the human ICAM1 promoter with a STAT3 binding site located at −115 to −107bp. The pGL3-ICAM1-SDM plasmid (bottom) with mutation in the STAT3 binding site as indicated. (B) HUVEC were transiently transfected via electroporation with pGL3-ICAM1-WT (Firefly), pRL-SV40 (Renilla) plasmids and different amounts of constitutively active STAT3 plasmid (1 μg and 3 μg) using Neon transfection system. Firefly and Renilla luminescence was measured and plotted as ratio. Mean ±SEM, two-tailed unpaired t-test. n=12 technical replicates. *P<0.05, ****P<0.0001. (C) Dual-luciferase assays were performed in HUVEC 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 ±SEM, two-tailed unpaired t-test. n=9 technical replicates. **P<0.01, ****P<0.0001. (D) Human VEGF-165 protein (25ng/ml) stimulated HUVEC lysates were immunoblotted for ICAM1, p-STAT3 (Y705), and total STAT3. (B–D) Depicted data is representative of multiple biological replicates. SDM: Site-directed mutagenesis.