Fig. 2

Modification of expression of ubiquitination genes in MSC-induced Tregs. CD4⁺CD25⁻ effector T cells and DCs isolated and cocultured with allogeneic MSCs under four conditions as described in Methods. Total mRNA extracted from MSC-induced Tregs after 6 h, 12 h, 24 h, 48 h, 72 h, and 5 days of coculture, and mRNA expression of TRAF6, GRAIL, USP7, UBC13, and STUB1 assessed by quantitative RT-PCR. Allogeneic MLR performed; CD4⁺CD25⁻ effector T cells isolated 5 days after MLR used as negative control, and TGF-β-induced Treg cells used as positive control. mRNA samples normalized to expression of endogenous housekeeping gene (GAPDH) and compared with negative control. Data presented as mean ± SEM; n = 4 independent experiments. Significant results: *p < 0.05; **p < 0.01; ***p < 0.001. Correlation of mRNA expression of each gene with that of FOXP3 mRNA shown under expression graph. Spearman correlation coefficient r and significance levels shown at top of graph. Significance levels of correlation coefficients: P*** (0.8 < CC < 1), P** (0.6 < CC < 0.8), and P* (0.4 < CC < 0.6); correlation coefficients less than 0.4 considered nonsignificant. A minus sign preceding correlation coefficient indicates negative correlation. d day, DC dendritic cell, iTreg induced Treg, LPS lipopolysaccharide, MSC mesenchymal stem cell, MSC-DC MSC-treated DC, MLR mixed lymphocyte reaction, ns not significant, TC allogeneic CD4⁺CD25⁻ T cells