Fig 2: NFATc2 expression correlates with EMT-related markers in melanoma. a Western blot analysis for NFATc2, ZEB1, SNAIL, N-cadherin, α-catulin, AXL, MITF, E-cadherin, ZEB2, and TWIST, in melanoma cell lines with the indicated genotype for BRAFV600E or NRASQ61R mutations. b Hierarchical clustering of log2-transformed and median-centered qPCR mRNA expression levels (2−ΔCt) for MITF, NFATc2, AXL, SNAI1, CDH2, ZEB1 genes in 30 melanoma cell lines with the indicated genotype for BRAFV600E or NRASQ61R mutations. c Spearman correlation analysis of mRNA expression values obtained by qPCR for the indicated genes in the panel of 30 melanoma cells lines shown in b. Positive and negative r-values are indicated in light blue and yellow, respectively.*p < 0.05; **p < 0.01; ***p < 0.001. d Spearman correlation analysis of the indicated genes in the TCGA melanoma dataset; r-values indicated as in c

Fig 3: Modulation of subcellular localization of NFATc2 by Zoledronic acid and anti-tumor effect of the combination of Zoledronic acid with GSK126 and of a GSK-3β inhibitor with GSK126. a Expression of NFATc2 by western blot analysis in cytoplasmic (C) or nuclear (N) fractions of melanoma cells treated or not with zoledronic acid (indicated as Zol, 150 μM, 72 h). b Anti-proliferative effect by MTT assay of Zoledronic acid (at three different doses in each cell line), GSK126 or their combinations on melanoma cell lines belonging to distinct mutational subset. c Apoptosis by annexin-V/PI assay at 72 h in melanoma cells treated with Zoledronic acid, GSK126 or their combinations. d Apoptosis by annexin-V/PI assay at 72 h in melanoma cells treated for 48 h with GSK-3β inhibitor AR-014418 (at 7.5 μM) and GSK126 (10 μM) or their combinations. Data from three-independent experiments. Statistical analysis in b, c, d by ANOVA and SNK post test. *p < 0.05; **p < 0.001, ***p < 0.001. Error bars indicate mean ± SD

Fig 4: Targeting of NFATc2, Myc, FOXM1, and EZH2 inhibits melanoma migratory and invasive activity. a Top panels: reduced melanoma cell migration in NFATc2 shRNA transfectants (NFATc2 shRNA_86a and NFATc2 shRNA_87a) compared to control transfectants (Ctrl_shRNA), evaluated by the wound closure assay. Bottom panels: representative images of the wound closure assay performed with NFATc2 shRNA transfectants and control transfectants. b Inhibition of Me71 melanoma migration by the wound closure assay by treatment for 48 h with inhibitors of NFATc2 (AM404), c-Myc (10058-F4), FOXM1 (Siomycin A), or EZH2 (GSK126). Results expressed as % closed wound area. c Inhibition of melanoma cell invasive activity, evaluated at 8 h by the CultrexCoat BME Cell Invasion Assay. Top panels: NFATc2 shRNA transfectants compared to control transfectants. Middle and bottom panels: melanoma cell line Me71 pre-treated for 48 h with the indicated inhibitors as in a. Statistical analysis in a, b, c by Student's t-test. ****p < 0.001. Error bars indicate mean ± SD. Data from four-independent experiments

Fig 5: Targeting of NFATc2 in melanoma cells downregulates c-Myc, FOXM1, and EZH2. a Expression by western blotting of NFATc2, c-Myc, FOXM1, EZH2, and H3K27me3 in three melanoma cell lines (Me71, Me79, Me98) at 72 h after transfection with two different NFATc2-specific Stealth siRNA (NFATc2 siRNA-1 and siRNA-2) or with control siRNA (ctrl siRNA). b, c Expression of NFATc2, c-Myc, FOXM1, EZH2, and H3K27me3 in b NFATc2 shRNA stable transfectants (NFATc2 shRNA_86a, and NFATc2 shRNA_87a) or in control cells (ctrl shRNA), or c in melanoma cell lines Me71, Me79, and Me98 treated or not for 144 h with the NFATc2 inhibitor AM404 (at 20 μM)