Fig 1: Nr4a Receptors Bind NFAT1, but Only Nr4a2 and Nr4a3 Are Calcineurin Pathway Dependent(A and B) Splenocytes from Tg4 Nr4a3-Tocky mice (A) or OTI Nr4a3-Tocky mice (B) were stimulated with 10 μM 4Y-MBP before analysis by flow cytometry for expression of Nr4a3-Timer Blue versus Nr4a3-Timer Red in live T cells.(C and D) Splenocytes from Tg4 Nr4a3-Tocky mice were stimulated with 10 μM 4Y-MBP (C) or OTI Nr4a3-Tocky were stimulated with 1 μM ova peptide (D) for 4 h in the presence of DMSO or 10 μM PP2 before analysis by flow cytometry for Nr4a3-Timer Blue versus Nr4a3-Timer Red expression in live CD4+ (C) or CD8+ (D) T cells.(E and F) Splenocytes from Tg4 Nr4a3-Tocky mice were stimulated with 10 μM 4Y-MBP variant for 4 h in the presence of DMSO, 1 μM cyclosporin A (CsA), or 1 μM FK506 before analysis of Nr4a3-Time Blue versus Nr4a3-Timer Red expression in live CD4+ Tg4 T cells by flow cytometry (E). Summary data showing the % Nr4a3-Timer Blue+ (F) or median Nr4a3-Timer Blue in live CD4+ Tg4 T cells (G); n = 4 (FK506) or n = 6 (unstim [unstimulated control], DMSO, and CsA).(F and G) Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test. Bars represent mean ± SEM; dots represent individual mice. *p<0.05, ***p<0.001, ****p<0.0001.(H–J) Splenocytes from OTI Nr4a3-Tocky mice were stimulated with 1 μM ova peptide for 4 h in the presence of DMSO, 1 μM CsA, or 1 μM FK506 before analysis of Nr4a3-Timer Blue versus Nr4a3-Timer Red expression in live CD8+ OTI T cells by flow cytometry (H). Summary data showing the % Nr4a3-Timer Blue+ (I) or median Nr4a3-Timer Blue (J) in live CD8+ OTI T cells, n = 4.(I and J) Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test. Bars represent mean ± SEM; dots represent individual mice. ****p<0.0001.(K) University of California, Santa Cruz (UCSC) genome browser tracks showing NFAT1 binding peaks in P14+Tcra−/− CD8+ T cells from GEO: GSE64409. UnstimKO, unstimulated NFAT1 knockout [KO]; Stim, phorbol myristate acetate (PMA) and ionomycin stimulation; StimKO, PMA and ionomycin stimulation in NFAT1KO mice.(L) Splenocytes from Tg4 Nr4a3-Tocky mice were stimulated with 5 μg/ml soluble anti-CD3 for 4 h in the presence of DMSO (−), 10 μM PP2, 1 μM CsA, or 1 μM FK506 before extraction of RNA and analysis of fold change in Nr4a receptor transcription; n = 3. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons test. Bars represent mean ± SEM; dots represent individual mice. *p<0.05, **p<0.01, ***p<0.001.(M) Splenocytes from Nr4a1-GFP Nr4a3-Tocky mice were stimulated with 5 μg/ml soluble anti-CD3 for 4 h in the presence of CsA or FK506. Live CD8+ T cells were analyzed by flow cytometry for expression of Nr4a1-GFP and Nr4a3-Timer Blue. See also Figure S1.