Fig 2: IRF4 inhibits NFATc1 and NFATc2 expression.(A) EBV positive or negative Akata BLs stably infected with pLKO (vector control) or IRF4 lentiviruses were treated with or without anti-IgG for 30 minutes and then harvested for immunoblot to detect expression of NFATc1, NFATc2, phospho-ERK, and tubulin loading control as indicated. (B) EBV-negative BJAB B cells were nucleofected with vector control, an LMP2A expression vector or an IRF4 expression vector as indicated then harvested after 48 hours for immunoblot analysis of the NFATc1, NFATc2, IRF4, LMP2A, and actin proteins. The numbers below the NFATc1 and NFATc2 immunoblots quantify the results using Image Studio Lite software to normalize the levels of NFATc1 and NFATc2 expression to actin expression. Results are presented as the ratio of NFATc1 and NFATc2 expression relative to actin for each condition, then averaged for each duplicate, and divided by the vector control values. Vector control values are set as 1.

Fig 3: NFAT1 expression is increased in spinal microglia after SNL. A) Real-time PCR shows the mRNA expression of NFAT1–4 after SNL. ** P < 0.01, *** P < 0.001, SNL versus Sham; Student's t-test, n = 5–6 mice per group. B) The time-course of Nfat1 mRNA expression in the ipsilateral dorsal horn in naïve, sham- and SNL-operated mice. * P < 0.05, ** P < 0.01, *** P < 0.001, SNL versus Sham. Student's t-test, n = 5–6 mice per group. C) Western blot shows the increased NFAT1 expression in WT mice after SNL from day 1 to day 21. * P < 0.05, ** P < 0.01, SNL versus Naive. Student's t-test, n = 3 mice per group. D) Immunostaining shows NFAT1 expression in the spinal cord of sham and SNL-operated (day 3 and day 10) mice. E–G) Representative images of double staining of NFAT1 with IBA-1 (E), GFAP (F), or NeuN (G). NFAT1 is highly colocalized with IBA-1. H,I) SNL-induced mechanical allodynia (F (1, 29) = 74.30, P < 0.0001, H) and heat hyperalgesia (F (1, 29) = 214.8, P < 0.0001, I) are markedly alleviated in Nfat1-/- mice compared with WT mice. *** P < 0.001, two-way RM ANOVA followed by Bonferroni's tests, n = 15–16 mice per group. J,K) Nfat1-cKO mice exhibit alleviated mechanical allodynia (F (1, 16) = 13.64, P = 0.002, J) and heat hyperalgesia (F (1, 16) = 64.75, P < 0.0001, K). * P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA followed by Bonferroni's multiple comparisons test, n = 9 mice per group.

Fig 4: NFATC2 is hypermethylated in patient fibroblasts. NFATC2 methylation in cells from patients with severe radiotherapy side-effects (n = 16) and controls (n = 8) were investigated on genome-wide methylation analysis. CpG sites in bold are differentially methylated between control and patient cells. CpG sites in red are part of promoter-associated regions defined by the ENCODE consortium.

Fig 5: Functionality of the CN–NFAT pathway in primary human monocytes. A) Calcium flux after ionomycin or curdlan (dectin-1 ligand) treatment of primary human monocytes. B and C) Fluorescence microscopy images of primary human monocytes showing NFAT1 translocation from the cytoplasm to the nucleus 45 min after treatment with Pam3CSK4 and zymosan or with ionomycine and zymosan in the presence or absence of cyclosporine A. The inhibitor was administered 60 min prior to the trigger. Quantification of nuclear translocation was performed using ImageJ software and a nuclear/cytoplasmic ratio plug-in