Skip to main content
. 2015 Dec;5(4):707–715. doi: 10.1086/684124

Figure 5.

Figure 5

Alteration of VEGF-induced permeability by Sp1 regulation of the nmMYLK promoter. A, Schematic depiction of the nmMYLK promoter, approximately 400 bp upstream of the transcription start site (TSS), with locations of potential transcription factor binding sites, including putative Sp1 binding sites (red bars), indicated. The promoter region analyzed for Sp1 binding by chromatin immunoprecipitation (ChIP) analysis is indicated by the green line. B, Quantitative polymerase chain reaction was performed with primers specific to the nmMYLK promoter region of interest, as indicated in A (green arrows). ChIP analysis using an Sp1 antibody demonstrated significant enrichment of the nmMYLK promoter sequence containing Sp1 binding sites from VEGF-treated (100 ng/mL, 24 hours) lung endothelial cells (ECs) compared with that in vehicle-treated cells (after normalization to the total chromatin input). ChIP analysis using a negative control immunoglobulin G (IgG) antibody resulted in negligible enrichment of the nmMYLK promoter sequence, with or without VEGF. Three independent experiments were performed, with bar graphs representing percent total chromatin input. *P < 0.05 versus vehicle. C, Confluent lung ECs treated with VEGF (100 ng/mL, 24 hours) showed a marked increase in nonmuscle isoform of myosin light chain kinase (nmMLCK) messenger RNA (mRNA) levels (compared with vehicle) that was attenuated with Sp1 silencing by means of small interfering RNA (siRNA). Three independent experiments were performed, with bar graphs representing fold change in pooled densitometry measurements. *P < 0.05 versus vehicle. D, Lung ECs were plated on gold microelectrodes for transendothelial electrical resistance (TER) measurements before Sp1 was silenced by means of siRNA, and then lung ECs were stimulated with vehicle or VEGF (100 ng/mL, 24 hours). VEGF-induced barrier disruption was attenuated by Sp1 siRNA (siSp1) but not by control siRNA (siCONT). Four independent TER experiments were performed, with pooled data represented as bar graphs. *P < 0.05 versus control siRNA. Inset shows Western blot demonstrating Sp1 protein silencing for C and D. HPRT: hypoxanthineguanine phosphoribosyltransferase; VEGF: vascular endothelial growth factor; Veh: vehicle.