adhesion was determined by Western blot using an anti-Y397 FAK mAb. As depicted in figure 8C, adhesion of control cells to Vn promoted FAK tyrosine phosphorylation on residue 397. Interestingly, SbS decreased the tyrosine phosphorylation level of FAK, confirming that SbS may regulate avb5 integrin activation (Figure 8C). SbS contains a heat-sensitive molecule, .9 kDa, that regulates both cell adhesion and migration We next sought to better characterize the active component(s) in SbS that blocked cell adhesion to Vn and induced cell migration. We first determined the heat stability of the active component. As illustrated in figure 9A, boiling the SbS for 10 min abrogated the inhibitory effect of SbS on cell-Vn adhesion. Similar results as those obtained for cell adhesion were observed for cell migration (Figure 9C), indicating that the blockade of both cell adhesion to Vn and induction of cell migration involved one or more proteins. We next examined the activity of SbS after passage through a 9 kDa cut-off filter. As illustrated in Figure 9B we found that the filtrate did not retain SbS activity in cell adhesion to Vn, clearly indicating that the active component(s) has a molecular weight Sb Modulates avb5 Integrin Activity higher than 9 kDa. Interestingly, although the filtrated fraction contained motogenic molecules, the flow-through filtration (,9 kDa) fraction was also able to increase cell migration. This indicates that SbS contains at least two molecules capable to modulate cell migration (Figure 9C). Discussion Intestinal epithelial restitution, proliferation and differentiation are all requirements for wound healing, a process disrupted in infectious or inflammatory bowel diseases, such as ulcerative colitis and Crohn’s disease [32]. Therefore, complete remission of such diseases requires both the cessation of inflammation and the repair of damaged epithelium. The development of novel therapies that accelerate the repair of intestinal epithelium has recently begun and various molecules are now being considered for clinical use. These include epidermal growth factor in combination with mesalamine [33], keratinocyte growth factor [34], and hepatocyte growth factor [35]. However, a better understanding of the biological effects of these molecules must be ascertained, not least to identify any undesired secondary effects, such as tumorigenesis. Clinical trials have suggested that Sb can be effective in the Figure 7. SbS alters the organization of adherence structures when cells are plated on vitronectin. HCT-8/E11 cells were plated on either fibronectin or vitronectin for 2 h and then fixed. av integrin (A) or vinculin (C) were detected using specific Abs. Focal adhesion labeling was quantified by measuring the area of each focal adhesion for all cells (B and D). Data represent the mean+SD of 3 separate experiments. *** P,0.001. doi:10.1371/journal.pone.0045047.g007 PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e45047