Fig 2: (A) The interaction between ESR1 and ß-catenin in HESCs was examined. Cells were incubated with DMSO and E2 (10-8 mol/l) for 24 h. Then cell lysates were prepared and immunoprecipitated with IgG and ESR1 antibody. The second panel is ESR1 shown as an internal loading control. Immunoblots of input lysate controls (5% of input) are also shown. (B and C) HESCs were incubated with E2 (10-8 mol/l) under different time scales (0, 12, 24, and 48 h). (B) Cell lysates were prepared and immunoprecipitated with ESR1 antibody or IgG. The interaction between ESR1 and ß-catenin/TCF3/LEF1 was examined. (C) Cell lysates were prepared and immunoprecipitated with ß-catenin antibody or IgG. The interaction between ß-catenin and ESR1/TCF3/LEF1 was examined. (D and E) Co-localization of ESR1, ß-catenin, and LEF1/TCF3 in nucleus were stimulated with E2 in HESCs. Cells were cultured in phenol red-free DMEM/F12 for 24 h and incubated with DMSO and E2 (10-8 mol/l) for 48 h. (D) Representative confocal microscopy images of HESCs immunostained for ERS1 (green, left panels) and its co-localization with the LEF1 and TCF3 (red, middle panels), and the merged images (right panels). (E) Representative confocal microscopy images of HESCs immunostained for ß-catenin (green, left panels) and its co-localization with the LEF1 and TCF3 (red, middle panels), and the merged images (right panels). The bars indicate 20 µm. Data presented are from three independent experiments.

Fig 3: (A) Schematic of the canonical Wnt signalling pathway and summary of key proteins analysed in this study. Proteins identified in the mass-spectrometry study are shown, those not analysed by Western blot are shown in grey. Western blot analysis of canonical Wnt components and GSK3ß protein expression characterization in cell models. 15 µg of total protein lysates from each cell-line were analyzed on a single phase 8% SDS gel and alpha-actin used as loading control. All were replicated in at least n = 3 unless indicated otherwise. Quantifications (arbitrary intensity units) are mean values ± SEM (Mann-Whitney U test p-value indicated). (B) Protein expression of GSK3ß and ß-catenin in HCT116 cells (independently sourced isolate), HCT116-GSK3ß-KO cells and HCT116-GSK3ß-WT cells. (C) ß-catenin, (D) TCF3 and TCF4, (E) Axin 2, (F) DVL2 and DVL3 (n = 2). All Westerns are representative examples, and quantifications are expressed as arbitrary intensity units. (G) TopFLASH reporter assay of canonical Wnt signalling activity in HCT116-GSK3ß-WT and HCT116-GSK3ß-KO cells. Cells were transfected with TopFLASH plasmids, TopFLASH luciferase activity was assessed 48 hours post-transfection following passive lysis of cells. Luciferase signaling was normalized to Renilla transfection control. p-value 0.064, n = 3.

Fig 4: mRNA relative expression of TCF-3 after transfection(A) The mRNA relative expression of TCF-3 in Eca-109 cells after transfection; (B) the silencing efficiency of siTCF-3-1 and siTCF-3-2 in Eca-109 cells; **, P<0.01 compared with the control group; #, P<0.01 compared with the siTCF-3-1 group.