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. 2020 Dec 23;218(2):e20201217. doi: 10.1084/jem.20201217

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© 2020 Wang et al.

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Figure 6. — ZFP91 restrains mTORC1 signaling in T reg cells. (A) IB analysis of the indicated proteins in Zfp91^+/+Foxp3-Cre and Zfp91^fl/flFoxp3-Cre T reg cells stimulated with antibodies against CD3 and CD28. (B) Quantifications of p-S6:GAPDH levels in Zfp91^+/+Foxp3-Cre (WT) and Zfp91^fl/flFoxp3-Cre (KO) T reg cells stimulated with anti-CD3 and anti-CD28 antibodies for 4 or 8 h are shown as the mean ± SEM of three experiments. (C and D) Flow cytometric analysis of p-S6 expression and Foxp3 expression in Zfp91^+/+Foxp3-Cre (WT) and Zfp91^fl/flFoxp3-Cre (KO) T reg cells stimulated with anti-CD3 and anti-CD28 antibodies for 4 h (n = 4 mice for each group). (E) Flow cytometric analysis of c-Myc expression in Zfp91^+/+Foxp3-Cre (WT) and Zfp91^fl/flFoxp3-Cre (KO) T reg cells stimulated with anti-CD3 and anti-CD28 antibodies in the presence of DMSO (Ctrl) or rapamycin (Rap) for 4 h (n = 4 mice for each group). (F and G) ECAR of Zfp91^+/+Foxp3-Cre (WT) and Zfp91^fl/flFoxp3-Cre (KO) T reg cells stimulated with anti-CD3 and anti-CD28 antibodies in the presence of DMSO (Ctrl) or rapamycin (Rap) for 3 h. (H and I) Flow cytometry analyzing Foxp3 expression (H) or cytokine expression (I) in Zfp91^+/+Foxp3-Cre (WT) and Zfp91^fl/flFoxp3-Cre (KO) T reg cells activated in vitro with anti-CD3, anti-CD28, and IL-2 for 24 h in the presence of DMSO (Ctrl) or rapamycin (Rap; n = 4 mice for each group). The data shown are representative of three independent experiments and are presented as mean ± SEM. ns, not statistically significant; **, P < 0.01. Two-tailed Student’s t test. 2-DG, 2-deoxy-D-glucose; MFI, mean fluorescence intensity; MW, molecular weight. mph, milli-pH units.