Figure 1
NFAT5 regulates Wnt/β-catenin signaling. (a) Effects of NFAT5 on TCF/β-catenin reporter activity. HEK293 and Caco-2 cells were co-transfected with TOPflash reporter plasmid or FOPflash-negative control plasmid together with either empty vector, Myc-NFAT5 or Flag-β-catenin alone or Myc-NFAT5 plus Flag-β-catenin, and the transfected cells were incubated for 48 h (upper panels). HEK293 and Caco-2 cells were transfected with control siRNA or siRNA targeting NFAT5. After a 48-h incubation, cells were transfected with TOPflash reporter plasmid or FOPflash-negative control plasmid. Twenty-four hours after retransfection, cells were incubated with control-conditioned medium or Wnt3a-conditioned medium for additional 12 h (middle panels). Cells were harvested and luciferase activity was measured in the crude cell lysates, as described in Materials and Methods. All results were normalized for transfection efficiency using the pRL-Tk-luc plasmid (Promega). Fold induction corresponds to luciferase activity of positive TOPflash reporter over negative FOPflash reporter. (Data represent mean±S.D.; *P<0.01 versus control vector or control siRNA; #P<0.01 s Flag-β-catenin alone or Wnt3a plus control siRNA, as determined by analysis of variance). The knockdown of NFAT5 was confirmed by western blot (lower panels). (b) Knockdown of NFAT5 increased mRNA expression of SP5 and c-myc, downstream targets of Wnt/β-catenin signaling. HEK293 and Caco-2 cells were transfected with control siRNA or siRNA targeting NFAT5. After a 48-h incubation, transfected cells were incubated with control-conditioned medium or Wnt3a-conditioned medium for additional 12 h. Total RNA was extracted, and SP5 and c-myc mRNA levels were determined by real time RT-PCR. (Data represent mean±S.D.; *P<0.01 versus control siRNA; #P<0.01 versus Wnt3a plus control siRNA, as determined by analysis of variance). (c) Overexpression of NFAT5 does not alter cytoplasmic or nuclear β-catenin levels. HEK293 cells were transfected with empty vector or Myc-NFAT5. After 48 h of transfection, cytosolic and nuclear proteins were extracted and expression of β-catenin was detected by western blot
