Fig 2: NFATc3-/- mice subjected to abdominal sepsis by CLP show healthy lung parametersNFATc3-/- and WT mice were subjected to CLP or sham surgery and analyzed after 24 h to determine (A) protein leakage in to BALF, (B). TNFa released in to BALF was measured by ELISA (C–D) Hema 3.0 Stained Neutrophils and macrophages in BALF. (E) NFATc3-/- and WT mice were subjected to LPS (i.p 15 mg/kg) sepsis for 16 h. Lungs were fixed, embedded in paraffin, sectioned and H&E stained. Lung histology showing neutrophilic infiltration in to NFATc3-/- and wild type mice. (A–D) *p = 0.001 WT-CLP vs WT- Sham; **p = 0.01 NFATc3-/- CLP vs WT-CLP; †p = 0.05 macrophages in NFATc3-/- CLP vs WT-CLP.

Fig 3: NFATc3-/- mice show improved arterial Oxygen saturation and survival compared to wild type mice(A) NFATc3-/- and wild type mice (N = 5) were injected with 15 mg/kg LPS i.p and the arterial oxygen saturation was measured using STARR Life Sciences Corp. Pulse Oximeter. (B–C) NFATc3-/- and wild type mice (N = 5 sham control group; N = 10 CLP group) were subjected to severe CLP, maintained on regular or imipenem monohydrate supplemented water and the survival was monitored over a period of 96 h. A* p = 0.001 WT-LPS vs. WT-Control; ** p = 0.01 NFATc3-/- LPS vs. WT-LPS.

Fig 4: NFAT does not appear to be involved in latent HIV transcription in 5A8 cells. A, immunoblotting analysis of RelA and NFAT knockdowns in 5A8 cells. Negative control siRNA (lanes 1 and 6), siRNAs against RelA (lanes 2 and 7), NFATc1, NFATc2, and NFATc3 (lanes 3 and 8), NFAT5 alone (lanes 4 and 9), and all four NFATs (lanes 5 and 10) were introduced into 5A8 cells by Amaxa nucleofection twice within 48 h. Cells were then treated with DMSO (lanes 1 to 5) or 500 nM CsA (lanes 6 to 10) for 2 h, stimulated with anti-CD3 and anti-CD28 antibodies for 24 h, and collected for immunoblotting or FACS analyses. CsA reduced the levels of NFATc1 and NFATc2, which autoamplified their expressions after TCR crosslinking. B, FACS analysis of LTR-driven GFP reporter activity (y-axis). Values are mean ± SEM from four experiments. Reporter activity remained partially sensitive to CsA inhibition (lanes 6 to 10) but was insensitive to NFAT knockdown (lanes 3 to 5). C, characterization of 5A8-NFAT-DsRed2 cells. Cells were pretreated with DMSO (panels 1–3) or 500 nM CsA for 2 h (panel 4) and incubated with 20 nM PMA (panel 2), or 20 nM PMA and 2 µM ionomycin (panels 3 and 4) for 24 h, followed by FACS analysis. PMA alone induced activity of the GFP reporter but not the DsRed2 reporter (panel 2). Ionomycin and PMA induced both reporters (panel 3). CsA fully inhibited combined PMA/ionomycin induction of DsRed2 reporter but only partially suppressed GFP reporter activity to levels similar to those after stimulation with PMA only (panel 4). D, effects of NFATc1, NFATc2 and NFATc3 knockdown on expression of NFAT-dependent DsRed2 and LTR-driven GFP. 5A8-NFAT-DsRed2 cells were nucleofected with negative control siRNA (panels 1 and 3) or siRNA against NFATc1, NFATc2, and NFATc3 (panels 2 and 4) twice within 48 h, treated with DMSO (panels 1 and 2) or 500 nM CsA (panels 3 and 4) for 2 h, and stimulated with anti-CD3 and anti-CD28 antibodies as in Figure 1. NFATc1, NFATc2 and NFATc3 knockdown decreased expression of the DsRed2 reporter but did not inhibit GFP reporter activity (panels 1 and 2). CsA partially reduced GFP reporter expression even in the absence of NFAT (panels 2 and 4).

Fig 5: Histone acetylation is more effective than DNA demethylation in reactivating gene expression in SET-accumulating cells(A) Quantitative real time PCR was performed using cDNA from HEK293, HEK293/SET, HN12, HN12siSET, HN13, and HN13siSET cells treated with 100 ng/mL TSA (histone deacetylase inhibitor) or 10 µM 5-aza-2'-deoxicytidine (DNA methyltransferase inhibitor). BRCA1, MLH1, and PTEN were assessed through TaqMan probes, and GSTP1, HIF-1A, NFATC3, and STAT1 through SybrGreen primers. Graphics represent relative quantification of experiments performed in triplicate, calculated using the 2-??Ct method. CDNA from HEK293 cells was used as calibrator; GAPDH and ß-globin primers were used as housekeeping. *(p < 0.05), **(p < 0.01), ***(p < 0.001) and ns (non-significant). (B) Transmission electron microscopy was performed in HEK293 and HEK293/SET cells to analyze chromatin compaction.