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. 2015 Sep 21;212(10):1623–1640. doi: 10.1084/jem.20142088

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© 2015 Sekiya et al.

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Figure 3. — Dysregulation of T reg cell–associated transcription in Nr4a-TKO T reg cells. (A) CpG methylation profiles of T reg cell–specific demethylated sites at the Foxp3 CNS2 enhancer. (B) Expression profiles of WT T reg cells (horizontal axis) and Nr4a-TKO T reg cells (vertical axis) of the indicated groups of genes. (C) qRT-PCR analysis of the indicated genes in WT and Nr4a-TKO T reg cells from 4–6-wk-old mice. Results are presented relative to expression of the control gene Hprt (n = 10 mice per genotype, pooled from 7 independent cohorts per genotype). (D) Flow cytometry profiles of CD4⁺ cells from the peripheral bloods of Rosa26^RFPCre-ER^T2-WT and Rosa26^RFPCre-ER^T2-Nr4a-TKO mice, before and after tamoxifen treatment. Numbers indicate the percentage of cells in the indicated regions. (E) Immunoblot analysis of Nr4a1 and Nr4a2 in sorted T reg cells (CD4⁺CD25^hiRFP⁺) from mice treated with tamoxifen, or in sorted T reg cells (CD4⁺CD25^hi) from littermates treated with vehicle. (F) qRT-PCR analysis of mRNA for the indicated genes in T reg cells (CD4⁺CD25^hiRFP⁺) from mice treated with tamoxifen, or in sorted T reg cells (CD4⁺CD25^hi) from littermate mice treated with vehicle. Results are presented relative to expression of the control gene Hprt (n = 6 mice per genotype, pooled from 2 independent cohorts per genotype). Error bars denote mean ± SD. *, P < 0.05; **, P < 0.01 (Mann-Whitney tests [C], one-way ANOVA with Bonferroni test [F]).