SbS modulates av integrin-mediated adhesion As regulation of cell adhesion and cell spreading are essential for cell migration, we next examined whether SbS altered HCT-8/ E11 cell attachment and spreading to both Fn and Vn. Vn is a ligand of avb5 integrin whereas Fn is recognized by a5b1 and avb3 integrins but not by avb5 integrin. However the primary sequence motif for the ligand binding of these integrins is a tripeptide RGD sequence [32]. Cells were either treated with SbS or not, then allowed to attach to increasing amounts of purified Fn or Vn. HCT-8/E11 cells attached to the two matrices tested (figure 3A). SbS did not alter cell adhesion to Fn. However, the percentage of cells adherent to Vn was dramatically decreased by SbS. As depicted by figure 3B, SbS down-modulated cell attachment to Vn in a dose-dependent manner, with a maximal effect obtained with a 1/8 dilution of SbS. Since cell spreading is initiated immediately after cell contact with matrix proteins, we also determined whether SbS affected cell spreading on both Fn and Vn. As observed in cell adhesion assays, SbS dramatically down-modulated the capacity of cells to spread on Vn. On the other hand, it weakly, albeit significantly, increased HCT-8/E11 cell spreading on Fn (Figure 3C). To delineate which a and b integrin subunits were affected by SbS during both cell adhesion and spreading to Vn, we performed adhesion assays in the presence of anti-integrin function-blocking Sb Modulates avb5 Integrin Activity mAbs. The interaction between HCT-8/E11 cells and Vn was mediated solely by the av integrin subunit (Figure 4A) whatever the condition tested. An anti- a5integrin subunit that blocked the HCT-8/E11 cell-Fn interaction (not shown) did not affect cell adhesion to Vn. It should be noted that when HCT-8/E11 cells were treated with SbS, blockade of the a5 integrin subunit led to a weak increase in cell adhesion to Vn (Figure 4A). This may suggest a functional interplay between av and a5 integrin subunits. As observed in Figure 4B, av subunit silencing also dramatically blocked cell adhesion to Vn, confirming that the av integrin subunit is the a subunit receptor for Vn in HCT-8/E11 cells. SbS competes with av integrin for binding to vitronectin Modulation of integrin expression at the cell surface may alter cell migration. To determine whether SbS modified integrin expression, we quantified the integrin subunits located at the plasma membrane by flow cytometry analysis. As shown in figure 5, SbS did not significantly alter expression levels of the av, b5, a6 and b1 integrin subunits. Since SbS inhibited cell adhesion to Vn without modulation of av integrin subunit expression, we postulated that it blocked av integrin-Vn interaction. To confirm this hypothesis, ECM-coated plates were incubated in the presence of SbS prior to addition of cells. We first determined that cells did not adhere to SbS coated plates (not shown). Addition of SbS to Vn-coated plates impaired cell adhesion (Figure 6A). Moreover, the same results were obtained when SbS-coated plates were incubated with Vn prior to cell adhesion (not shown). However, when the same experiments were performed using Fn instead of Vn, no change in cell attachment was found (Figure 6A). This suggests that SbS specifically interacts with Vn and blocks av-dependent HCT-8/ E11 adhesion. Furthermore, similar results were observed using another intestinal cell line HT29-D4 (Figure 6B), indicating that the observed phenomenon could be common to intestinal cell lines. Figure 4. avb5 integrin is required for HCT-8/E11 cell interac- tion with vitronectin. (A) Isolated HCT-8/E11 cells were incubated without (Ctrl) or with SbS (SbS) in the absence (-) or presence of either anti-av or -a5 integrin mAbs. Cells were then seeded on vitronectin (3,15 mg/ml). Cell adhesion 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) HCT-8/E11 cells were transfected with anti -av-integrin siRNAs (SiRNAav) or scramble oligos (SiRNACtrl) for 48 h. Cells were then plated on vitronectin for 2 h. Cell-ECM 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.g004 Figure 5. Impact of SbS on integrin expression. Sub-confluent HCT-8/E11 cells were incubated for 5 h with (dark bars) or without (light bars) SbS, then harvested and resuspended in the presence of anti- integrin subunit mAbs. After incubation with the appropriate secondary Alexa 488-conjugated Ab, cell-bound fluorescence was quantified using a Becton-Dickinson FACScan flow cytometer. Non-specific labeling was determined by incubating cells with the secondary Alexa 488- conjugated Ab alone (Ctrl). Data represent the mean+SD of 6 separate experiments. doi:10.1371/journal.pone.0045047.g005 PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e45047