specificity of these observations, we evaluated the impact of Sc supernatant on cell migration. As illustrated in Figure 1C, the supernatant of Sc did not modulate wound repair. Furthermore, similar results as those obtained with Sb supernatants in HCT-8/ E11 cells were observed using other polarized intestinal cell lines including HT29-D4, T84 and CaCo2/TC7 (Figure 1D), indicat- ing that the observed phenomenon could be common to all intestinal cell lines. We verified by flow cytometry analysis that T84 cells express both a2b1and av integrins indicating that the reduced effect of Sb in this cell line was not associated with a lack of integrin expression (not shown). Sb-induced cell restitution is independent of cell proliferation To determine whether the enhanced wound repair observed in response to Sb supernatant was caused by an increase in cell migration and/or cell proliferation, HCT-8/E11 cells were treated with 2.5 mg/ml mitomycin C, an inhibitor of DNA replication, both before and during wound healing assays. Mitomycin C did not attenuate the Sb-dependent wound repair, indicating that Sb acts by increasing epithelial cell migration with no effect on proliferation (Figure 2A). Furthermore, as a marker of cell proliferation rate, the extent of BrdU incorporation into cells was measured. As shown in Figure 2B, few nuclei of migrating cells exhibited uptake of BrdU. Moreover, no significant difference between untreated or Sb-treated cells was observed. Taken together, these findings indicate that Sb supernatant improves intestinal epithelial restitution indepen- dently of cell proliferation. S. boulardii Enhances Cell Restitution Sb supernatant improves enterocyte crypt-villus migration To evaluate whether Sb supernatant regulates enterocyte migration in vivo, C57BL6J mice were force-fed with PBS solution with or without 1 mg/ml Sb, daily, for 1 week. This treatment did not modify villus length (not shown). BrdU was administrated to control and Sb force-fed mice 24 hours before death. This strategy allowed the calculation of the rate, and extent to which BrdU- labelled cells migrated from the crypts to the tips of the villi [26]. In control animals, BrdU-stained cells were detected mainly within the crypts (Figure 3A). By contrast, in Sb force-fed mice, BrdU positive cells were distributed from the crypt to the tips of the villi (Figure 3B). Statistical analysis revealed that Sb significantly enhanced cell migration along the crypt-villus axis (migration to 45% of villus height in Sb treated animals, compared to 20% of villus height in controls) (Figure 3C). To ensure that Sb did not promote the expansion of the proliferative compartment, ki-67, a marker of proliferating cells, was immunolocalized. As depicted in Figures 3D and 3E, ki-67 positive cells were detected only within the crypts in both control and Sb force-fed mice. Moreover, Sb treatment did not modulate the number of proliferating cells in the crypts (Figure 3F). These finding indicate that Sb could modulate enterocyte migration, both in vitro and in vivo, without affecting the proliferative compartment. To compare the effect of the whole yeast and the yeast supernatant, we daily administrated to the mice for one week, either the whole yeast either the culture supernatant of S.boulardii. A group of mice were also force-fed daily with supernatant of S. Cerevisiae. As depicted on Figures 4A and 4C, BrdU-stained cells Figure 2. Sb-stimulated cell restitution is independent of cell proliferation. (A) HCT-8/E11 cell monolayers were incubated for 2 hours with (+) or without (2) 2.5 mg/mL mitomycin C, wounded and cultured for an additional 5 hours period with or without mitomycin C, in the absence or presence of Sb supernatant. Wound closure was evaluated as described in Methods. Results are expressed as the percentage of cell migration compared to control. Data represent the mean 6 SD of 3 separate experiments. * P,.05. (B) Confluent monolayers were wounded and incubated with or without Sb supernatant for 4 hours. BrdU (10 mmol/L) was added and incubation continued for an additional hour. Cells that had incorporated BrdU into their DNA were detected as described in Methods and are indicated by arrowheads. Data shown are from a representative experiment out of 3 performed. Scale bar: 300 mm. doi:10.1371/journal.pone.0018427.g002 PLoS ONE | www.plosone.org 4 March 2011 | Volume 6 | Issue 3 | e18427