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. 2014 Jun 23;111(27):E2807–E2816. doi: 10.1073/pnas.1401505111

Fig. 4.

Fig. 4.

MeCP2 is required for the maintenance of Foxp3 expression in Tregs during inflammatory cytokine stimulation in vitro. (A–C) CD4+CD25+GFP+ Tregs from LNs and spleens of Mecp2f/y Foxp3–GFP–Cre or their WT littermate control mice were sorted, labeled with the CellTrace proliferation dye, and stimulated with 1 μg/mL plate-bound anti-CD3 and 1 μg/mL plate-bound anti-CD28 in the presence of 50 U/mL IL-2 with or without 50 ng/mL IL-6 for 5–7 d (n = 3). (A) The percentages of viable cells retaining CD25 and Foxp3 expression at the indicated time points were measured by flow cytometry. Data show means ± SEM. (B) The percentages of viable cells at the indicated time points were determined by LIVE/DEAD Fixable Violet Dead Cell Staining. Data show means ± SEM. (C) Proliferation of viable cells at the indicated time points were determined by dilution of the CellTrace proliferation dye. Data show means ± SEM. (D) Viable WT and MeCP2-deficient Treg cells after 5 d of culture with anti-CD3/CD28, IL-2, and IL-6 were sorted, and expression of Treg signature genes was determined by qPCR. Data show means ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.