Fig 2: NFAT factors are major downstream effectors of calcineurin in T-ALL.(A) Schematic of the experiment: leukemic cells obtained from an ICN1-induced T-ALL carrying 2 floxed alleles of CnB1 (T-ALL #3) were retrovirally transduced with MIG vectors encoding either HA-tagged CnB1, or HA-tagged NFAT1*, or HA-tagged NFAT1*-NLS, or the control vector without insert (MIG), co-cultured on MS5 stromal cells and immediately treated with 4OHT to delete CnB1 floxed alleles. (B) PCR genotyping analysis for the floxed and deleted alleles of CnB1 in cultured cells 2 days after 4OHT treatment. PCR for Hsp70 is used as control. (C) Left panel: Expansion of CnB1-deleted leukemic cells transduced with MIG (black squares), MIG-CnB1 (blue triangle), MIG-NFAT1* (red triangles), MIG-NFAT1*-NLS (orange); expansion over time is reported as the cumulative cell numbers over 17 days in MS5 co-cultures. Right panel: in the same co-cultures, enrichment in GFP+, transduced cells, was followed over time. This experiment is representative of three independent experiments. (D) PCR genotyping analysis for the floxed and deleted alleles of CnB1 in the GFP+ (transduced) and GFP- (non-transduced) fractions of flow-cytometry sorted leukemic cells from the respective co-cultures at day 17. PCR for Hsp70 is used as control.

Fig 3: 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 4: Functional redundancy of NFAT factors in T-ALL.(A) Schematic description of the experiment: mice were injected with leukemic cells obtained from either primary T-ALL #68 (ICN1; RCT2; Nfat1-/-; Nfat2fl/fl; Nfat4+/+) or #14 (ICN1; RCT2; Nfat1-/-; Nfat2+/+; Nfat4fl/fl), that carry wild type alleles of Nfat4 or Nfat2, respectively. When BM leukemia burden reached about 10–15% T-ALL cells in these recipients, mice received 3 successive daily injection of either carrier solvent (So, n = 3) or Tamoxifen (TAM, n = 6) to induce Nfat2 (T-ALL #68) or Nfat4 (T-ALL #14) floxed alleles deletion thus resulting in T-ALLs relying upon only Nfat4 (T-ALL #68) or only Nfat 2 (T-ALL #14). Mice from all groups became terminally leukemic 2 days later, sacrificed and Nfat-proficient (blue label, 1) or Nfat-defloxed (red label, 2) cells were flow cytometry-sorted and compared for their ability to re-initiate leukemia in secondary recipient mice under limit dilution conditions. Flow cytometry-sorted leukemic cells obtained from donor mice (1, 2) and retrieved from terminally ill recipients (1’, 2’) were genotyped for Nfat floxed and deleted alleles. (B) Left panels: PCR genotyping of Nfat2 floxed and deleted alleles in input leukemic cells from T-ALL #68 (1 and 2; see schematic in A) and in leukemic cells recovered from one representative secondary recipient injected T-ALL cells (T-ALL #68, 2’). Right panels: PCR genotyping of Nfat4 floxed and deleted alleles in input leukemic cells from T-ALL #14 (1 and 2; see schematic in A) and in leukemic cells recovered from one representative secondary recipient injected with T-ALL #14 (2’). PCR for Hsp70 is used as control. (C) Left panel: Kaplan-Meier survival curves of mice transplanted with T-ALL cells #68 expressing only Nfat4 (red tracing) or co-expressing Nfat2 and Nfat4 (blue tracing). Right panel: Kaplan-Meier survival curves of mice transplanted with T-ALL cells #14 expressing only Nfat2 (red tracing) or co-expressing Nfat2 and Nfat4 (blue tracing).

Fig 5: 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