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Supporting Information Table 1. List of primers used for qRT-PCR.

Supporting Information Figure 1. MSC recruitment of Treg cells. (Ai-Ci) Representative plots showing the percentage of CD4+CD25high, CD4+FOXP3+,CD25+FOXP3+ cells after 4 day's CD4+/MSCs co-culture in the absence or presence of MSCs. (Aii-Bii) Graphic representation of flow cytometric data derived from at least five independent experiments (mean ± SD) *p < 0.05, **p < 0.01 ***p < 0.001. (D) qRT-PCR analysis of FOXP3 mRNA, was analysed from cells prepared under the same conditions as in panel Ai. The data was normalized to GAPDH and represented as fold change with respect to levels of control cells. Data derived from five independent experiments (mean ± SD); **p < 0.01. (E) Graphic representation of flow cytometric GITR expression after 4 day's CD4+/MSCs co-culture in the absence or presence of MSCs, data derived from four independent experiments (mean ± SD)*p < 0.05 Data were analyzed by unpaired t-test using Prism3 Software (GraphPad Software).

Supporting Information Figure 2. TGF-®1 is not involved in MSC-mediated Treg cells induction. (Ai-Ci) Representative plots showing the percentage of CD4+CD25+, CD4+CD25high, CD4+FOXP3+, CD4+CD25+FOXP3+ and CD4+CD25highFOXP3+ cells after 4 day's CD4+/MSCs co-culture in presence of TGF-®1 neutralizing antibody. Isotype antibody was used as controls (CTRL). (Aii-Cii) Graphic representation of flow cytometric data derived from at least three independent experiments (mean ± SD). (D) qRT-PCR analysis of FOXP3 mRNA, was analysed from CD4/MSCs co-cultures in the presence of TGF−®1 neutralizing antibody. Isotype antibody was used as controls (CTRL). The data was normalized to GAPDH and represented as fold change with respect to levels of control cells. Data derived from at least four independent experiments (mean ± SD).

Supporting Information Figure 3. GSI-I treatment no effect on MSC-Notch signaling pathway. (A) qRT-PCR analysis of Notch1, and Notch1-ligands mRNA (Jagged1, Jagged2, DLL1, DLL3, DLL4) on MSCs treated with GSI-I compared with untreated MSCs as control. The data was normalized to GAPDH and represented as fold change with respect to levels of control cells Data derived from three independent experiments (mean ± SD).

Supporting Information Figure 4 Western blot assays of Jagged1 expression on MSCs. Representative western blot analysis of Jagged1 expression in MSCs. GAPDH was used as loading control.

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