Supplemental Materials

This article contains the following supporting material:

• Supplementary Figure 1 - Overview and optimization of the permeability assay system. (A) Schematic representation of the transwell assay system. To mimic the endothelium in vivo, we plated the EC on matrigel-coated 3-µm pore size filters in the transwell inserts. In the final optimized system, the cells were plated twice, as described in the Material and Methods section, to ensure the formation of a tight hMVEC monolayer. As a permeability indicator, we used 3kDa FITC-dextran (5µg/ml) which was introduced into the lower chamber along with the permeability-inducing agent. The permeability was quantified as fluorescence units of the FITC that passed from the lower chamber into the upper chamber, and was plotted over time after addition of the reagents. (B) Effect of initial cell plating number on the endothelial barrier function. Number of cells plated/well are indicated in the graph. The filters were pre-coated with matrigel and the hMVEC cultured for 48 hours prior to initiating the permeability assay. Cells plated twice were plated at 24-hour intervals. Data represent mean +/- the standard error. Statistics are shown as comparisons of the group plated twice with 1 x 10⁵ cells each time with all other groups, *P<0.05. (C) The system was tested using uncoated transwell inserts, matrigel-coated inserts, or a tight monolayer of hMVEC on top of matrigel-coated inserts. Data represent mean +/- the standard error. Statistics are shown as comparisons of the insert only and insert coated with matrigel groups with the hMVEC monolayer on top of matrigel-coated inserts, **P<0.01. (D) The transwell filters were coated with collagen type I, fibronectin, or matrigel prior to cell plating as described in Materials and Methods. Data represent the mean +/- the standard error. Statistics are shown as comparisons of the cells plated on matrigel with those plated on firbonectin or collagen I, *P<0.05, **P<0.01.
• Supplementary Figure 2 - IL-8 increases the levels of a tyrosine phosphorylated band corresponding to VEGFR2. Confluent HMEC-1 were treated with 100 ng/ml IL-8 for various time points, with cell extracts analyzed by immunoblot analysis with a phospho-tyrosine antibody (upper panel). Extracts were also analyzed for total VEGFR2 content to identify the tyrosine phosphorylated band as VEGFR2 and to provide a loading control (lower panel). The phospho-tyrosine band corresponding to VEGFR2 also increased in a biphasic manner, with the early phase initiated by 30s after treatment.
• Supplementary Figure 3 - IL-8-induced Src phosphorylation is blocked with the Src inhibitor SU6656. Confluent HMEC-1 were treated with 100 ng/ml IL-8 for 1 min., +/- pre-incubation with μM SU6656 for 5 min. Proteins were extracted, and equal protein concentrations were separated by SDS-PAGE and subjected to immunoblot analysis using an antibody that specifically recognizes Src phosphorylated at tyrosine 419, indicative of active Src (upper panel). The blot was stripped and re-probed with an antibody that recognizes total Src to determine equal loading (lower panel). IL-8-induced Src phosphorylation was blocked by the Src kinase inhibitor SU6656.