Supplementary Figure 5: Metabolic and epigenetic analyses of Atg7-deficient T_reg cells.

(a) Measurement of OCR in T_reg cells (from Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice) stimulated with anti-CD3 and anti-CD28 for 4 h. (b) Methylation status of CpG motifs of the TSDR at Foxp3 locus, assessed by bisulfite sequencing of divided T_reg cells (from Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice) activated with anti-CD3, anti-CD28 and IL-2 for 96 h in the presence of DMSO or DCA. Numbers above boxes (1-14) indicate the 14 CpG islands from 5′ to 3′ in the intron 1 of the Foxp3 locus. (c) Immunoblot analysis of HK2 in T_reg cells (from Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice) stimulated with anti-CD3 and anti-CD28 for 4 h. (d) Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl mice received mock or rapamycin treatment (n=4 mice per group). T_reg cells were sorted and stimulated with anti-CD3 and anti-CD28 for 4 h, followed by analysis of mRNA expression of Hk2. (e,f) mRNA expression (e) and immunoblot analysis (f) of HIF1α in Foxp3^CreAtg7^+/fl and Foxp3^CreAtg7^fl/fl T_reg cells stimulated with anti-CD3 and anti-CD28 for 4 h. NS, not significant (P > 0.05); * P < 0.05 (two-tail unpaired Student's t-test in a,e and one-way ANOVA in d). Data are representative of two (a) or three (b,c,f) experiments, or pooled from four out of four (d,e) experiments (mean ± s.e.m in a,d,e).