Constitutive activation of SFKs through expression of DN-Csk renders HDMEC susceptible to low doses of TNF-α.

<p>Cells were seeded at confluence and incubated for 72 hours. Then, growth media was replaced by low serum media and cells were infected with adenovirus to express either LacZ (control) or DN-Csk. After another 16 h, cells were treated with low-dose TNF-α. <b><i>A</i></b>, Western blot analysis demonstrating the increased tyrosine phosphorylation at multiple SFK substrates. <b><i>B</i></b>, TEER measurements on ECIS electrodes. <b><i>C</i></b>, FITC-Albumin permeability assay on Transwell chambers. <b><i>D</i></b>, TEER of cells pretreated with either DMSO or PP2 for 30 minutes prior to TNF-α addition. Results are representative of at least three independent experiments. Notice that the synergistic effect of DN-Csk expression concurrently with a low-dose TNF-α can be prevented by the SFK inhibitor PP2. Data presented as mean±SEM (each experiment performed in duplicate). Asterisk, p<0.05 (Two-way ANOVA and Tukey’s multiple comparison post-hoc test). Bar graphs with data presented as mean±SEM and statistical analysis of TEER for the 20 h time point corresponding to B and D are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161975#pone.0161975.s006" target="_blank">S6C and S6D Fig</a> respectively.