Knock-down of av integrin by small interfering RNA av integrin suppression in HCT-8/E11 cells was performed as previously described [30]. Statistical analysis Unless noted, data are presented as the means 6 S.D. for three independent experiments performed in triplicate. Comparison between the two conditions was made by using the Mann– Whitney test for sampling,30. P,0.05 was considered statistically significant in all analyses and is indicated by ‘***’ when ,0.001, ‘**’ when ,0.01 and ‘*’ when ,0.05. Results avb5 integrin supports SbS-mediated cell migration We previously reported both in vitro and in vivo that SbS improves intestinal cell restitution through activation of the a2b1 integrin collagen receptor [15]. However it is clear that a2b1 integrin is not the sole receptor involved in this process since a function-blocking anti-av subunit integrin mAb partially inhibited SbS-mediated cell migration (figure 1 and supplemental video S1 and S2). Quantification of the surface area recovered by the cells indicated that the anti- av subunit integrin mAb inhibited the migration Sb Modulates avb5 Integrin Activity capacity of SbS-treated HCT-8/E11 cells by approximately 70% (figure 1). To delineate which avb integrin was involved in the migratory process we incubated HCT-8/E11 cells with function- blocking anti-avb3, anti-avb5or anti-avb6integrin mAbs during cell migration. As observed in figure 1B, only the anti- avb5integrin mAb partially blocked both control and SbS-induced HCT-8/E11 cell migration. avb5 integrin is redistributed in SbS force-fed mice Since avb5 integrin is involved in SbS-induced cell migration in vitro, we next evaluated whether SbS affected the distribution of avb5 integrin in vivo. C57BL6J mice were force-fed daily with either RPMI 1640 or SbS, for 1 week. We verified that SbS treatment did not alter the mucosal architecture. The distribution of b5 integrin subunit was determined by immunohistochemistry on intestinal tissues (figure 2). In control mice, b5 subunit was abundantly detected at the surface of cells located in the crypts and along the apical membrane domain of cell present in the villus. By contrast, in SbS force-fed mice, b5 positive cells were mainly redistributed to the basal surfaces of enterocytes in the villus (figure 2B). The pattern of E-cadherin expression was also analyzed along the crypt-villus axis in order to locate epithelial cells. As observed on figure 2, E-cadherin positive cells also Figure 2. avb5 integrin is relocalized in the intestine of SbS force-fed mice. Mice were daily force-fed for one week with unused culture medium for Sb (Ctrl) or SbS (SbS). Frozen sections of small intestinal tissues were co-stained with a rabbit anti-b5-b integrin subunit (b5) and a rat anti E-cadherin (E-cad). Sections were then incubated with a mixture of Alexa 594-conjugated and Alexa 488-conjugated secondary antibodies against rabbit and mouse IgG, respectively. Rabbit Ig and rat Ig correspond to isotype controls Abs. Colocalized pixels appear in yellow in merged images. Each image is a representative image taken from tissue sections of four mice. The arrow and the arrowhead point out a villus and a crypt, respectively. doi:10.1371/journal.pone.0045047.g002 PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45047