Regulation of c-Rel by IkBe in Murine Lymphocytes Figure 1. TNF inhibits IL-2 gene transcription and attenuates induction of AP-1 and NFAT. 11A2 cells were cultured with or without TNF 2.5 ng/ml for 8 days before (A) restimulation with plate-bound anti-CD3e 5 mg/ml, PMA 10 ng/ml and ionomycin 50 ng/ml (P+I)low, or PMA 50 ng/ml and ionomycin 100 ng/ml (P+I)high for 24 hours. Secreted IL-2 protein was measured by immunoassay (mean +/2 SEM, n = 5 experiments); (B) restimulation as above for 4 hours followed by RNA extraction, and analysis for IL-2 mRNA by RNase protection assay (mean +/2 SD, n = 3 experiments). After restimulation with (P+I)low, nuclear and cytoplasmic extracts were prepared, and equivalent amounts of protein assayed by immunoblot for (C) nuclear AP-1 and (D) nuclear NFAT2 (representative of 3 experiments for each condition). Each blot was probed then reprobed for the indicated proteins. doi:10.1371/journal.pone.0024504.g001 detected consistently by immunoblot. In resting primary T cells, hyper-phosphorylated NFAT is bound to inactive calcineurin phosphatase in the cytoplasm. A sustained calcium signal activates calcineurin, leading to dephosphorylation and nuclear transloca- tion of NFAT. NFAT1 is constitutively expressed in T cells whereas NFAT2 is inducible. In 11A2 cells, dephosphorylated NFAT1 was expressed constitutively in the nucleus, to similar levels in control and TNF-treated cells (Figure S2). Induced nuclear NFAT2 was detected at two hours, with levels increasing after 4 hours’ stimulation (Figure 1D). Its nuclear expression was lower and slightly delayed relative to control cells, but NFAT2 was nonetheless strongly induced, dephosphorylated and imported to the nucleus in TNF-treated cells. TNF pre-treatment inhibits subsequent induction and nuclear expression of NF-kB associated with upregulation of IkBe p65 and c-Rel were detected in the cytosols of resting control and TNF-treated 11A2 cells (Figure 2A, panels 1, 3). Newly- synthesised c-Rel appeared in the cytosol of control and, to a lesser extent, TNF-treated cells after 1–2 hours’ stimulation with PMA and ionomycin (Figure 2A, panel 1). However, nuclear c-Rel, detectable at 2 hours, was greatly reduced in TNF-treated relative to control cell samples (panel 2) and remained barely detectable at 6 or 8 hours (not shown). TNF-treated cells also showed a reduction in nuclear translocation of both p65 (panel 4) and p50, more p50 being retained in the cytosol of TNF-treated compared to control cells (panels 5, 6). To test whether the inhibitory effects of TNF on NF-kB signalling were limited to the TCR pathway, whose activation is mimicked by PMA and ionomycin, we analysed nuclear and cytosolic NF-kB in control and TNF-pre-treated 11A2 cells following acute restimulation with TNF (50 ng/ml). Acute TNF stimulation led to rapid, transient nuclear translocation of c-Rel, p65 and p50 which peaked between 30 minutes and 1 hour of stimulation (Figure 2B, panels 2, 4, 6). However, c-Rel was barely detectable in nuclear extracts of TNF-pre- treated cells at any time-point of acute TNF stimulation (panel 2). By contrast, acute TNF-induced nuclear translocation of p65 and p50 was only slightly reduced at most time-points tested (panels 4, 6), although more p50 remained in the cytosol of TNF-pre-treated cells PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24504