Inhibition of MEK, which is known to activate ERK1/2, blocks VCAM-1-dependent spleen leukocyte TEM.

<p>Confluent monolayers of mHEVa cells were nontreated (NT) or treated for 30 minutes with the MEK inhibitors PD98059 (20 µM) or U0126 (20–40 µM). To block leukocyte binding to VCAM-1, the endothelial cells were treated with a blocking anti-VCAM-1 antibody without a secondary crosslinking antibody. <b>A–D</b>) Splenic leukocytes were added to the endothelial monolayer, allowed to briefly settle to mediate cell contact and then exposed to 2 dynes/cm² laminar flow for 15 minutes to examine migration (<b>A and B</b>) or 2 minutes to examine leukocyte-endothelial cell association (<b>C and D</b>). Then, cells were washed and fixed in 3% paraformaldehyde for 1 hour and examined by phase contrast microscopy <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-Deem1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-AbdalaValencia1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-Ager1" target="_blank">[65]</a>. Non-migrated leukocytes are phase-light and migrated leukocytes appear as phase-dark <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-Deem1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-AbdalaValencia1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-Ager1" target="_blank">[65]</a>. We previously reported that the leukocytes that migrated are >88% lymphocytes as determined by flow cytometry <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-Tudor1" target="_blank">[6]</a>. <b>E</b>) Relative cytotoxicity was determined by the G6PDH assay; PD98059 (20 µM) and U0126 (40 µM) were not cytotoxic as compared to the nontreated control cells. Data in each panel are from 3 experiments. *, p<0.05 compared to <b>A,C</b>) NT groups, <b>B,D</b>) 0 minutes groups, or compared to DMSO-treated or last washes (data not shown). Inhibitors had no effect on cell viability, as determined by Trypan blue exclusion (data not shown).