Figure 2.

Effects of S27D on viability on endothelial cells. (A) TNF-α-stimulated HUVEC were treated for 30 minutes with S27D (5μg/mL), control peptide (CTRL) or DMSO and subsequently trypsinized, fixed and stained with Annexin-V-FITC and Propidium iodide (PI). Plots show that after S27D treatment, 77% of the cells were negative for Annexin-V. After DMSO and control peptide (CTRL) treatment, respectively 89% and 88% of the cells were negative. All cells were negative for PI, showing that the treatment did not induce necrosis. Neutrophils (PMN) were left overnight (O/N) and used as positive Annexin-V control. Only 15% of the PMNs were Annexin-V negative. (B) Effect of S27D on apoptosis of endothelial cells at different time points are analyzed using Annexin-V-FITC labeling. Experiment is carried out three times. Data are mean ± SEM. (C) HUVEC were treated as described under A. Expression of ICAM-1, VCAM-1, PECAM-1 and VE-cadherin was measured by flow cytometry analysis and expressed as Mean fluorescent Intensity (MFI) arbitrary units on the Y-axis. S27D was incubated for different time points as indicated. VE-cadherin expression is increased after overnight treatment with S27D. Experiment was carried out three times in triplicate. Data are mean ± SEM. *p<0.05.