Sb Modulates avb5 Integrin Activity SbS regulates the avb5 integrin signaling pathway Activation of avb5 integrin leads to remodeling of the actin cytoskeleton through the formation and activation of a large signaling complex called focal adhesion. This adhesion complex contains enzymes and scaffolding molecules including FAK and vinculin [32]. Since SbS modulated HCT-8/E11 adhesion to Vn, we first checked whether SbS regulated avb5 integrin activation. We first analyzed the impact of SbS on the organization of adherence structures after plating cells on Vn. As depicted by Figure 7A, av integrin subunit staining revealed that control cells plated on Vn, exhibited focal adhesion structures. However, av integrin subunit staining in SbS treated cells displayed less focal adhesion structures. Analysis of the av integrin subunit detected at the cell-substratum interface revealed that SbS markedly decreased the area of focal adhesion sites (Figure 7B). The same results were obtained with vinculin (Figures 7C and 7D). These results indicate that treatment with SbS is associated with structural modifications of focal adhesion structures. Interestingly this effect was not observed when Fn was used as a substrate (Figures 7A–B and 7C– D). We next analyzed whether SbS the avb5-mediated signaling pathway. We first determined whether SbS affected protein tyrosine phosphorylation by immunolocalization. In untreated cells, the mAb PY20 directed against the phosphorylated tyrosine residue, mainly stained cell-ECM contact sites, namely focal adhesion structures (Figure 8A). However, SbS treatment promot- ed a decrease in the area of the tyrosine phosphorylated focal adhesion sites (Figures 8A and 8B). This suggests that SbS may negatively modulate the activity of focal adhesion structures. To confirm the impact of SbS on the avb5 signaling pathway, cells were either treated with SbS or not and plated on a Vn-coated surface for 1 h. The tyrosine phosphorylation status of FAK post- Figure 6. SbS blocked avb5 integrin interaction with vitronectin. (A) Vitronectin- or Fibronectin-coated plates were incubated in the absence (Vitronectin and Fibronectin) or presence of SbS (vitronectin+ SbS and fibronectin+ SbS) prior to addition of cells. Isolated HCT-8/E11 cells incubated without (light bars) or with SbS (dark bars) were then seeded on plates. Cell adhesion to ECM was evaluated as described in Methods. Results are expressed as the percentage of cell adhesion compared to untreated cells (Ctrl). Data represent the mean + SD of 3 separate experiments. (B) Isolated HT29-D4 cells were treated with or without SbS (dilution 1/8) and plated on vitronectin at the indicated concentrations. Cell-vitronectin adhesion was evaluated as described in Methods. Results are expressed as the percentage of cell adhesion. Data represent the mean+SD of 3 separate experiments. *** P,0.001. doi:10.1371/journal.pone.0045047.g006 PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e45047