Fig 2: NFAT3/RERG interaction is increased in luminal breast cancer tissues and is correlated with the absence of ALN colonization. (A) Frozen luminal breast cancer sections from patients who developed ALN colonization (N+; a-f) or not (N0; g-l) were incubated with a-NFAT3 and a-RERG followed by PLA assays (yellow dots) and labeling with a-pan cytokeratin coupled to FITC. Data from 6 representative patient slides out of 21 are shown. (B) Index of PLA was obtained by analyzing 2 random fields for the 21 slides; data are shown as mean ± SEM (**p<0,005). (C) Characteristics of the patients used for microscopy.

Fig 3: NFAT3-Cter acts as a dominant negative form of endogenous NFAT3. (A) T-47D cells were transiently transfected with either a vector encoding the HA epitope fused to NFAT3-Cter or with the empty vector (Vector). The following day, transfected cells were subjected to an in vitro invasion assay for 24 h. All data are shown as the mean of three independent experiments ± SEM (n=3 biological replicates; 3 technical replicates for each experiment; *p<0.005). Whole cell lysates were revealed by a-HA and normalized by revelation with a-actin. (B) T-47D cells were transiently cotransfected with either an siRNA control (siCtl) or an siRNA targeting the endogenous NFAT3 (siNFAT3) in combination with either a vector encoding the HA epitope fused to NFAT3-Cter or with the empty vector (Vector). Approximately 48 h later, transfected cells were subjected to an in vitro invasion assay for 24 h. The mean of three independent experiments ± SEM is shown (n=3 biological replicates; 3 technical replicates for each experiment; *p<0,05, ***p<0,001, ns, non-specific). Whole cell lysates were revealed by a-HA and a-NFAT3 and normalized by revelation with a-actin.

Fig 4: The last 85 C-terminal amino acids of NFAT3 are required for the inhibition of cell invasion. (A) Schematic representation of the homologous and isotype-specific regions of human NFAT1 and NFAT3 with the activation domains (ADs), the NFAT homology domain (NHR), the Rel homology domain (RHR), and the DNA-binding domain (DBD). (B) Schematic representation of the NFAT3, NFAT3-85C, ?NFAT3, and ?NFAT3-85C constructs used in the study and the sequence of the last 85 amino acids of NFAT3 (NFAT3-Cter). (C) T-47D cells were transiently transfected with either a vector encoding the HA epitope fused to NFAT3, NFAT3-85C, ?NFAT3, or ?NFAT3-85C or with the empty vector (Vector). The following day, transfected cells were subjected to an in vitro invasion assay for 24 h. All data are shown as the mean of two independent experiments ± SEM (n=2 biological replicates; 3 technical replicates for each experiment, **p<0.005, ***p< 0.0001, ns, non specific relative to the vector-transfected cells). Whole cell lysates were revealed by a-HA and normalized by revelation with an a-actin.

Fig 5: NFAT2 is markedly increased in human renal tissue with severe fibrosis.Immunostaining for NFAT1, NFAT2, NFAT3 and NFAT4 was performed on tissues from IgAN patients and adjacent normal tissues from renal cell carcinoma patients. Representative confocal images showed higher expression and more nuclear localization of NFAT2 in the renal tubules, interstitium and glomerulus in IgAN patients with severe renal fibrosis (n = 3) than in IgAN patients with mild renal fibrosis or in adjacent normal renal tissue from renal cell carcinoma patients (n = 3) (Scale bars = 50 µm). Masson staining was used to assess kidney fibrosis (scale bars = 100 µm). a Immunostaining for NFAT1 and NFAT3. b Immunostaining for NFAT2 and NFAT4. c–f Quantitative analysis for the immunofluorescence of NFAT1, NFAT2, NFAT3 and NFAT4. *P < 0.05 vs. Control; #P < 0.05 vs. IgAN with mild renal fibrosis. NFAT1 nuclear factor of activated T cells 1, NFAT2 nuclear factor of activated T cells 2, NFAT3 nuclear factor of activated T cells 3, NFAT4 nuclear factor of activated T cells 4.