Stretch of Septic Monolayers Figure 1. BODIPY staining increases with stretch magnitude. The area (m 6 SE) stained by BODIPY-conjugated ouabain was determined in sham and 2CLP monolayers following 1 hour of cyclic stretch (0.25 Hz) to 0% (NS), 12%, or 25% DSA (N$3 isolations). Area stained in sham monolayers did not increase above unstretched levels, while stained areas in 2CLP monolayers significantly increased above unstretched control levels following stretch to both magnitudes. Confocal micrographs of Na+/K+-ATPase pumps on the basal membrane of unstretched and stretched (1 hour, 25% DSA) 4-day old sham and 2CLP monolayers demonstrated that increases in BODIPY staining was not due to differences in receptor expression (Scale bar in Sham – NS = 100 mm). Significant differences between stretch groups were determined by Tukey tests. Significance defined as p,0.01, bar indicates significant difference between sham and 2CLP at same stretch magnitude, *significantly different than 2CLP unstretched. (mean 6 SE). doi:10.1371/journal.pone.0038748.g001 Phosphorylation of MAPk Proteins Phosphorylation of JNK, ERK, and p38 MAPk was analyzed after 0, 10, and 60 minutes of stretch to 12% and 25% DSA. As reported previously, in unstretched monolayers JNK and ERK phosphoryla- tion was significantly increased in 2CLP monolayers compared to sham (bar, Figure 2) [15]. Stretch of 2CLP and sham monolayers to 12% DSA did not produce significant increases in JNK, ERK, or p38 phosphorylation (Figure 2). We concluded that activation of MAPk signaling was not responsible for the increased permeability of 2CLP monolayers following 60 minutes of stretch to 12% DSA. A larger stretch magnitude of 25% DSA resulted in significant increases in the phosphorylation of JNK and ERK in sham monolayers compared to unstretched sham controls (Figure S1), and significant increases in the phosphorylation of ERK in 2CLP monolayers compared to unstretched sham and 2CLP controls. Phosphorylation of p38 was not observed in 2CLP or sham monolayers at either stretch magnitude (data not shown). Tight Junction Protein Expression The transmembrane proteins of the tight junction, including claudins 3, 4, 5, 7, 8, and 18, occludin, and JAM-A, are ultimately responsible for regulating paracellular permeability, and therefore we analyzed their expression levels following stretch. As reported previously [15], we again observed significantly reduced expres- sion of claudin 4, claudin 18, and occludin levels in whole cell lysates from unstretched 2CLP monolayers compared to sham (NS bars, Figure 3). Stretch to a magnitude of 12% DSA did not significantly alter the expression levels of any of the TJ proteins probed in 2CLP monolayers (Figure 3). Low magnitude stretch significantly reduced only the expression of claudin 7 in sham monolayers compared to unstretched. Therefore we concluded that loss of tight junction expression in 2CLP monolayers was not responsible for the observed permeability increases at low stretch magnitudes. Interestingly, we did observe decreases in claudin 7 and ZO-1 expression in sham monolayers following high stretch magnitudes (Figure 3). Inhibition of ERK phosphorylation during high magnitude stretch with U0126 had no effect on permeability or tight junction expression in 2CLP monolayers (Figures S2 and S3). In contrast, ERK inhibition in sham monolayers prevented stretch-induced changes in permeability and in Z01 protein expression. PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e38748