PBS, cells were incubated with Alexa Fluor 488-conjugated sheep Ig (20 mg/ml), raised against mouse and rabbit Igs, for 1 h, then washed and mounted in ProLong GoldH (Invitrogen). Images were captured and analyzed using a SP5 Leica confocal microscope equipped with LAS AF Lite software. Focal adhesion (FA) labeling with anti-vinculin, -avb5 and -phosphotyrosine Abs was quantified using ImageJ software by measuring the area of each focal adhesion for all cells. Approximately 40 cells for each condition were analyzed. Immunohistochemistry Groups of 6-week old C57BL6J female mice (n = 4) were force- fed daily with 200 mL RPMI 1640 or with 200 mL SbS, for 1 week. After the sacrifice of mice, segments of intestine were frozen in liquid nitrogen and cryosectioning (section thickness: 8 mm) was performed. Samples were fixed in acetone (220uC for 10 min), rehydrated then blocked for 30 min in PBS containing 4% (w/v) BSA. avb5 integrin and E-cadherin were detected by an overnight incubation at 4uC with a rabbit anti-b5 integrin subunit (clone H- 96; Santa Cruz Biotechnology) and a rat anti-E-cadherin mAb (clone ECCD-2, Millipore), respectively. After 3 washes in PBS, cells were incubated with Alexa Fluor 594- or 488-conjugated sheep Ig (20 mg/ml), raised against rabbit and rat Igs respectively, for 1 h, then washed and mounted in ProLong GoldH (Invitrogen). Non-specific labeling was determined by incubating cells with a mixture of rabbit Igs and rat Igs followed by an incubation with the secondary Alexa 594-and 488-conjugated Abs. Images were captured and analyzed using a SP5 Leica confocal microscope equipped with LAS AF Lite software. All animal experiments were performed in accordance with the regulations of our institution’s ethics commission. They were conducted following the APS Guiding Principles in the Care and Use of Animals. The study was approved by the Ethics Committee in Animal Experimentation of Centre Me´diterrane´en de Me´decine Mole´culaire (C3M), Nice, France (protocol 3/2010). Cell migration assay Monolayers of differentiated HCT-8/E11 cells were wounded using a sterile tooth-pick and incubated with or without various dilutions of SbS. Plates were placed in a temperature and CO2- controlled chamber mounted on a Nikon TE2000 inverted microscope. Images were captured every 5 min for a total observation period of 5 h using a Cool SnapHQ camera (Princeton Instruments) through a 106 objective lens. For each wound, 10 measurements of wound width were recorded. To assess the role of the integrins in SbS enhanced enterocyte migration, 10 mg/ml of the following function-blocking anti- integrin mAbs were used: av integrin (69.6.5, Beckman Coulter), avb3integrin (mouse IgG1, LM 609 clone, Millipore), avb5inte- grin (mouse IgG1, P1F6 clone, Millipore), or avb6 (mouse IgG2a, 10D5 clone, Millipore). Rat-anti dipeptidyl peptidase IV (clone 5H2, gift of S. Maroux, CNRS, Marseille, [28]), and mouse anti- a5 subunit (mouse IgG2, clone SAM-1; Millipore) were used as isotypic Abs controls. Abs were added both 1 h before wounding and during the period of cell migration. Detection of tyrosine-phosphorylated FAK HCT-8/E11 cell suspensions (800,000 cells/1 ml), prepared in adhesion buffer, were seeded onto Vn- or Fn-coated wells and allowed to adhere for 1 h. Both adherent cells and cells in suspension were lyzed as previously published [29]. Equal amounts of cell lysates (25 mg) were resolved by SDS-PAGE and blotted onto a nitrocellulose sheet. Membranes were blocked with PBS containing 4% BSA and probed overnight at 4uC with a Sb Modulates avb5 Integrin Activity mouse anti-Y397-FAK (Invitrogen). Blots were then revealed by chemiluminescence after incubation with the appropriate horse- radish peroxidase-conjugated secondary Ab (Amersham). Loading amounts were verified by probing the blot with a rabbit-anti FAK (Ozyme) Ab or with a rabbit-anti-b tubulin (Sigma) Ab. Figure 1. avb5 integrin is required for SbS-stimulated cell restitution. (A) Polarized HCT-8/E11 cell monolayers were pretreated without or with 10 mg/ml of an anti- av integrin mAb (anti-av) for 1 h. Cell monolayers were then wounded as described in Methods and incubated for 5 h in the absence (ctrl) or presence of SbS in a medium either containing anti-av integrin mAb or not. Phase contrast images were acquired at the indicated times. Data shown are from a representative experiment out of 3 performed. Scale bar: 300 mm. (B) HCT-8/E11 cell monolayers were wounded and incubated with SbS. The monolayers were further incubated without (-) or with the following function-blocking anti-mAbs 1 h before wounding and during cell migration: avintegrin (av), avb3 integrin (avb3), avb5 integrin (avb5), or avb6 integrin (avb6). Wound closure was determined as described in Methods. Results are expressed as the percentage of cell migration compared to SbS-treated cells without mAbs. Data represent the mean+SD of 5 separate experiments. Rat-anti DPP IV (DPPIV) and mouse anti-a5 subunit (a5) were used as isotypic Abs controls. *** P,0.001. doi:10.1371/journal.pone.0045047.g001 PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45047