Inhibition of IFN-I responses by NFAT5. (A) GSEA of microarray data comparing wild-type (Nfat5+/+, WT) and NFAT5-deficient (Nfat5−/−, KO) BMDMs stimulated with poly(I:C) (pIC,1 µg/ml, 6 h). NFAT5-deficient macrophages showed a significantly enhanced IFNγ response signature upon poly(I:C) stimulation (top), but not an unrelated response (apoptosis Hallmark signature, bottom). (B) Volcano plot analysis representing the distribution of Interferome database genes differentially expressed between Nfat5+/+ and Nfat5−/− BMDMs stimulated with poly(I:C) (1 µg/ml, 6 h). Out of 2,731 genes differentially expressed between Nfat5+/+ and Nfat5−/− BMDMs (shown above the P < 0.05 significance dashed line), 1,209 corresponded to IFN-I–regulated genes annotated in the Interferome database, of which 758 genes were upregulated in Nfat5−/− BMDMs (blue dots) and 451 in wild-type BMDMs (red dots). See Table S1 for the list of genes. (C and D) Western blot analysis of NFAT5 expression after poly(I:C)- or IFNα-stimulation in wild-type BMDMs (two independent experiments are shown in C) and in IFNAR-deficient (Ifnar1−/−) and control (Ifnar1+/+) BMDMs (D) for the indicated times. Results in the graphs show the mean ± SEM of three independent experiments after normalization with pyruvate kinase (PyrK) expression. Statistical significance in C and D was determined with a one-sample t test using NFAT5 expression in unstimulated cells as reference with a value of 1. *, P < 0.05; **, P < 0.01. (E) Western blot analysis for phospho-STAT1 (Y701) and STAT1 in Nfat5+/+ and Nfat5−/− BMDMs left unstimulated or stimulated with poly(I:C), as indicated. These results are complementary to Fig. 1 I.
Figure S1.

Inhibition of IFN-I responses by NFAT5. (A) GSEA of microarray data comparing wild-type (Nfat5+/+, WT) and NFAT5-deficient (Nfat5−/−, KO) BMDMs stimulated with poly(I:C) (pIC,1 µg/ml, 6 h). NFAT5-deficient macrophages showed a significantly enhanced IFNγ response signature upon poly(I:C) stimulation (top), but not an unrelated response (apoptosis Hallmark signature, bottom). (B) Volcano plot analysis representing the distribution of Interferome database genes differentially expressed between Nfat5+/+ and Nfat5−/− BMDMs stimulated with poly(I:C) (1 µg/ml, 6 h). Out of 2,731 genes differentially expressed between Nfat5+/+ and Nfat5−/− BMDMs (shown above the P < 0.05 significance dashed line), 1,209 corresponded to IFN-I–regulated genes annotated in the Interferome database, of which 758 genes were upregulated in Nfat5−/− BMDMs (blue dots) and 451 in wild-type BMDMs (red dots). See Table S1 for the list of genes. (C and D) Western blot analysis of NFAT5 expression after poly(I:C)- or IFNα-stimulation in wild-type BMDMs (two independent experiments are shown in C) and in IFNAR-deficient (Ifnar1−/−) and control (Ifnar1+/+) BMDMs (D) for the indicated times. Results in the graphs show the mean ± SEM of three independent experiments after normalization with pyruvate kinase (PyrK) expression. Statistical significance in C and D was determined with a one-sample t test using NFAT5 expression in unstimulated cells as reference with a value of 1. *, P < 0.05; **, P < 0.01. (E) Western blot analysis for phospho-STAT1 (Y701) and STAT1 in Nfat5+/+ and Nfat5−/− BMDMs left unstimulated or stimulated with poly(I:C), as indicated. These results are complementary to Fig. 1 I.

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