Fig. 4.

TGF-β induces Twist transcriptionally upregulating Wnt3 and leads to activate Wnt/β-catenin pathway. a top E-box located in promoter of Wnt3 where Twist binding allowed; bottom Cells were treated with or without TGF-β and ChiP-qPCR assay was performed as described in “Methods” section. The bars indicate the fold enrichment of Twist binding to the promoter of Wnt3 in the indicated cells with or without TGF-β induction and compared to Mock cells (immunoprecipitated with IgG). b The cells were treated with shRNA Twist or negative sequence for 48 h. RT-qPCR was conducted with Twist primer and the bars in left indicated relative expression of Twist (mean ± SD from three determinations) adjusted with 18S, *p < 0.05 compared to untreated cells. In the right, Western blot was performed with antibodies specific for Twist and Wnt3. The β-actin was used for loading control. c The cells were treated with Twist shRNA or negative control sequence and then induced with or without TGF-β. The nuclear or cytoplasm expressions of β-catenin were determined by Western blot analysis. The α-Tubulin and H3 were used for loading control of cytoplasm and nuclear protein respectively. d The bar graph indicates the regulation of Wnt/β-catenin pathway signaling in SKBR3 wild type (blue bars), wild-type cells treated with TGF-β (red bars) and shRNA Twist knocking down cells (black and green bars) treated with TGF-β compared to untreated SKBR3 (blue bars) analyzed by specific Wnt/β-catenin mRNA array. The relative level of each indicated gene was adjusted with GAPDH and each bar indicates mean fold change and SD from four determinations