Fig 2: NFATc2 is a c-Rel target gene in TRAIL-resistant Panc1 cells. (a) Hierarchical clustering of top 50 differentially expressed genes in Panc1 cells and MiaPaca2 cells. Transcripts were selected for being differentially expressed in either of the cell lines and associated with characterized genes. Transcripts are displayed in rows and samples are listed in columns, whereas the row dendogram represents the similarity between the individual transcripts. Transcription levels and experimental groups are colour coded. Hierarchical clustering was performed using the unweight pair group method with arithmetic mean as a cluster algorithm, and similarities between transcripts are based on their Spearman rank correlation. For better readability, all transcripts were z-score normalized before cluster analysis. (b) Transcript levels were normalized to unstimulated cells and the 12 genes with the highest fold induction in the resistant Panc1 cells transfected with the control siRNA (black columns) are presented in comparison with the sensitive MiaPaca2 cell line transfected with the control siRNA (white columns). Data represent the mean of three independent experiments. (c) Transcript levels were normalized to unstimulated cells and the effect of the transfection of resistant Panc1 cells with the c-Rel siRNA in comparison with the resistant Panc1 cells transfected with the control siRNA are presented. Data represent the mean of three rindependent experiments. *P-values <0.05 for the effect of the c-Rel siRNA on TRAIL-mediated gene induction

Fig 3: Characterization of a functional c-Rel binding site in the NFATc2 promoter. (a) Schematic representation of the putative c-Rel binding site (MatInspector query). For comparison, the c-Rel/p50 consensus site GGRRNNYYC (where R is for A/G, Y for C/T and N for any base) is shown. The putative binding motive in the NFATc2 promoter is highlighted in bold. (b and c) Panc1 (b) or Patu8998t cells (c) were left untreated (withe columns) or treated with 10 ng/ml TRAIL for 3 h (black columns) and qChIP were conducted with IgG, anti-c-Rel, anti-NFATc2 and anti-Rbp1 antibodies. Data are expressed as enrichment relative to IgG levels and represent the mean values±S.D. from six independent experiments performed in duplicates. *P-values <0.05

Fig 4: Physical interaction between NR2F6 and NFATc1 is dependent on DBD and LBD domains of NR2F6. Unstimulated or PMA/Iono-stimulated Jurkat cells were cotransfected with wildtype and mutant NR2F6-His, Strep-HA tagged NFATc1 and the Il17a promoter region (1.3 kb) constructs, and nuclear protein extracts were pulled down with Streptavidin beads. (A) Anti-Strep immunoprecipitates of the total nuclear lysates were immunoblotted with anti-His and anti-HA antibodies, which revealed direct binding of NR2F6-NFATc1 in resting but not PMA/Iono-stimulated cells. The addition of ethidium bromide destroyed the interaction between NR2F6 and NFATc1 in resting cells. (B) DNA (C112S; S89E) and ligand binding domain (LL/AA: E383Stop) mutants of NR2F6 lost their affinity to bind NFATc1 or NFATc2 in resting Jurkat cells, in contrast to wt-NR2F6. The input and GFP-negative controls are shown, along with the DNA polymerase loading control. One of three independent experiments is shown.

Fig 5: TRAIL-induced NFATc2 expression and DNA binding are c-Rel dependent. (a and b) Panc1 (a) and Patu8998t (b) cells were transfected with the indicated siRNA for 48 h. Then cells were left untreated or treated with 10 ng/ml TRAIL for 3 or 5 h. Total RNA was submitted to reverse transcription and real-time PCR detecting NFATc2. For normalization, beta-actin was analysed as control. Data are expressed as normalized mRNA level and represent the mean values±S.D. from four independent experiments performed in duplicates. *P-values <0.05. (c) Patu8998t (left part) and Panc1 (right part) cells were left untreated or were treated with 10 ng/ml TRAIL for the indicated time periods and submitted to EMSA for measurement of NFAT-binding activity. A representative of four independent experiments is shown. (d) Nuclear extracts from Panc1 (upper part) or Patu8998t cells (lower part) treated for 5 h with 10 ng/ml TRAIL were submitted to EMSA with the indicated antibodies and was performed using the indicated oligonucleotides. A representative of four independent experiments is shown. (e) Panc1 (upper part) and Patu8998t (lower part) cells were transfected with the indicated siRNA (set1) for 48 h, and then treated with 10 ng/ml TRAIL for 5 h and submitted to EMSA for measurement of NFAT-binding activity. A representative of four independent experiments is shown