Figure 2.

MENK reduced the viability of CD4+CD25+ Treg cells and inhibited TGF-β mediated conversion in vitro. (A), The viability of CD4+CD25+Treg cells was assessed by MTS. (B), Isolated CD4+CD25-T cells underwent TGF-βconversion in the presence of plate-coated anti-CD3 plus soluble anti-CD28 and IL-2 for 48 h or 72 h. The cells receiving treatment of MENK at 10⁻¹² M or RPMI 1640 alone were examined by FCM analysis. Gate was set on CD4+CD25+, and the expression of Foxp3 was shown. (C), Real-time PCR was conducted to quantify the mRNA level of Foxp3 of the cells receiving treatment for 72 h. (D), FCM analysis for the expression pattern of CTLA-4, CCR4, GITR, and CD69 in TGF-βinduced Treg cells for 72 h. Data was investigated by Flowjo software. The red curve was the isotype control staining; the blue curve indicated specific staining. Data was collected from at least 2 independent experiments with similar results and presented as the mean±SD (triplicates). *P < 0.05; **P < 0.01 versus that in RPMI 1640 group (CONT group) as determined by Student's t test or one-way ANOVA.