Existence of a nuclear NFATc1–STAT3 complex in pancreatic cancer.

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Existence of a nuclear NFATc1–STAT3 complex in pancreatic cancer. Existence of a nuclear NFATc1–STAT3 complex in pancreatic cancer. A, genome-wide expression and GSEA analysis in p48-Cre;KrasG12D; Nfatc1 tumor cells. Negative normalized enrichment score (NES) indicates loss of gene enrichment upon NFATc1 knockdown (additional information in Supplementary Table S1). B, heatmap showing selection of differentially regulated genes in p48-Cre;KrasG12D;Nfatc1 tumor cells depending on NFATc1 expression. Fold change relative to control cells is displayed in a blue–white–red pseudo color scheme for selected genes with FClog2 < 1.5 or FClog2 > −1.5. STAT3 expression changes are highlighted in red (details in Supplementary Table S2). C, qRT-PCR displaying Stat3 expression upon NFATc1 depletion in p48-Cre;KrasG12D;Nfatc1–derived tumor cell clones. D and E, pancreatic lysates from p48-Cre;KrasG12D;Nfatc1 and p48-Cre;KrasG12D mice were assessed for Stat3 mRNA expression (D) or total STAT3 protein expression and phosphorylation of STAT3 at Y705 [pSTAT3 (Y705); E]. F and G, immunohistochemical analysis for STAT3 and pSTAT3 (Y705) in p48-Cre;KrasG12D;Nfatc1 mice tumors (F) and NFATc1 and pSTAT3 (Y705) in human PDA (G). Scale bars, 100 μm. H, statistical illustration of tissue microarray (TMA) analysis (n = 215 patients) demonstrating high correlative expression levels of nuclear NFATc1 and pSTAT3 in human PDA tissues. I, immunofluorescence staining displays intracellular localization of STAT3 (green) and NFATc1 (red) in p48-Cre;KrasG12D;Nfatc1 tumor cells. Nuclei are visualized by Hoechst staining (blue). J, coimmunoprecipitation of endogenous NFATc1 and STAT3 was performed in murine KrasG12D;Trp53−/− PDA cells and human Panc1 cells upon TGFβ and IL6 treatment. K and L, coimmunoprecipitation for NFATc1 and STAT3 in p48-Cre;KrasG12D;Nfatc1–derived cells transfected with FLAG-tagged wild-type (wt)-STAT3 and (K) FLAG-STAT3 (Y705F) or (L) treated with 1 μmol/L WP1066 for 3 hours [blocking STAT3 (Y705) phosphorylation]. Sandra Baumgart et al. Cancer Discovery 2014;4:688-701 ©2014 by American Association for Cancer Research