|
Fig. 2 (A) Localization of EIF4A3 in HeLa cells, visualized by immunofluorescence (red) with an anti-EIF4A3 antibody. Nuclei (blue) were counterstained with DAPI. Scale bar: 20 μm. (B) EIF4A3 directly binds to β-catenin and TCF7L2. (C,D) Mapping of the region in the TCF7L2 responsible for the EIF4A3 interaction. Schematic diagram of human TCF7L2 protein domains is shown in C. Various HA-tagged TCF7L2 deletion mutants were co-expressed with Flag-tagged EIF4A3 in HEK293T cells and cell lysates were subjected to co-immunoprecipitation (D). Asterisks indicate specific bands. (E) Endogenous EIF4A3 is associated with the promoters of the indicated Wnt target genes in HEK293T cells, as indicated by the ChIP assay. The promoters of GAPDH and α-Satellite were used as negative controls. (F) EIF4A3 represses β-catenin/TCF7L2-mediated transcriptional activity. (G) EIF4A3 impairs the interaction between β-catenin and TCF7L2. Left panel: HCT116 cells were co-transfected with tagged β-catenin and TCF7L2 along with various doses of EIF4A3. Right panel: EIF4A3 at various doses was transfected into HCT116 cells. Proteins were extracted from cell lysates, immunoprecipitated and subjected to a western blot analysis using the indicated antibodies. (H) BIO treatment releases EIF4A3 from the indicated Wnt target genes in HEK293T cells, as indicated by ChIP assay. HEK293T cells were treated with DMSO or 1 μM BIO for 4 h and then cells were harvested for the ChIP assay. The promoter of GAPDH was used as a negative control. WRE, Wnt response element; ORF, open reading frame; PRO, promoter region. (I-K) BIO treatment decreases the EIF4A3 levels in the β-catenin and TCF7L2 immunoprecipitant, and a similar effect is seen on β-catenin and TCF7L2 levels in the EIF4A3 immunoprecipitant. HEK293T cells were treated with DMSO or 1 μM BIO for 4 h; cells were then harvested and proteins were extracted from cell lysates, immunoprecipitated and subjected to a western blot analysis using the indicated antibodies to determine the interaction among EIF4A3, β-catenin and TCF7L2 under different Wnt activity. (L) Working model for the role of EIF4A3 involved in the β-catenin/TCF complex. Data are mean±s.e.m. (n=3). *P<0.05; **P<0.01; ***P<0.001, ns, not significant. Unpaired t-test, two-tailed.