S. boulardii Enhances Cell Restitution type I collagen-coated wells and allowed to adhere for periods ranging from 0 to 30 minutes. Adherent cells were lyzed as previously published [24]. Equal amounts of cell lysates (50 mg) were resolved by SDS-PAGE and blotted onto a nitrocellulose sheet. Membranes were blocked with PBS containing 5% non-fat dry milk and probed overnight at 4uC with Abs directed against rabbit phospho ERK1/2 (Ozyme, St. Quentin en Yvelines, France), mouse anti-Y397-FAK (Invitrogen) or rabbit anti-Y118-paxillin (Millipore). Blots were then revealed by chemiluminescence after incubation with the appropriate horseradish peroxidase-conjugated secondary Ab (Amersham). Loading amounts were verified by probing the blot with rabbit-anti FAK (Ozyme), mouse anti-Erk1/ Erk2 (Santa Cruz Biotechnology), or mouse anti-paxillin (Millipore). Statistical analysis All experiments were performed a minimum of 3 times in duplicate. For statistical analysis of data, Student’s t-test was used. Values are expressed as mean 6 SD. Data were considered as statistically significant at P values of ,.01(**) or ,.05 (*). Results Sb supernatant improves colonic epithelial cell restitution To investigate any direct effect of Sb supernatant on cell restitution, we used HCT-8/E11 cells for their wound-filling capacity [25]. In vitro, Sb supernatant markedly enhanced wound repair (Figure 1A) in a dose-dependent manner (not shown) with a maximal effect obtained with a 1/8 dilution. We therefore used this dilution in subsequent experiments. Time lapse videomicro- scopy experiments (see supplemental video S1 and S2) revealed that cells treated with Sb supernatant exhibited a higher rate of migration than cells treated with a unused culture medium for Sb (80 mm/h versus 40 mm/h). Quantification of the surface area recovered by the cells indicated that Sb supernatant exerted its effect as early as 1 hour after wounding and persisted for up to 5 hours (Figure 1B). The motogenic effect of Sb supernatant was quite similar to that observed using type I insulin-like growth factor (IGF-I), a well known inductor of cell restitution (Figure 1C) [24]. To confirm the Figure 1. Sb increases intestinal epithelial cell restitution. (A) Polarized HCT-8/E11 cell monolayers were wounded as described in Methods and incubated for 5 hours with or without Sb supernatant. Phase contrast images were acquired at the indicated times (see also Supplementary data S1 and S2). Data shown are from a representative experiment out of 5 performed. Scale bar: 300 mm. (B) HCT-8/E11 cell monolayers were wounded and incubated with or without Sb supernatant. The percentage of wound area closed was determined at hourly intervals. Data represent the mean 6 SD of 3 separate experiments. ** P,.01. (C) HCT-8/E11 cell monolayers were wounded and incubated for 5 hours without (control) or with Sb or Sc supernatants, or 14 nM IGF-I (IGF-I). Results are expressed as the percentage of cell migration compared to control. Data represent the mean 6 SD of 3 separate experiments. ** P,.01. (D) Polarized HT29-D4, CaCo2/TC7 and T84 cell monolayers were wounded and incubated for either 4 hours (T84) or 20 hours (HT29-D4 and CaCO2/TC7) with or without Sb supernatant. Results are expressed as the percentage of cell migration compared to control. Data represent the mean 6 SD of 3 separate experiments. ** P,.01. doi:10.1371/journal.pone.0018427.g001 PLoS ONE | www.plosone.org 3 March 2011 | Volume 6 | Issue 3 | e18427