Supplementary Figure 1. HEK293 Were Transiently Transfected with The
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Supplementary Figure 1. HEK293 were transiently transfected with the indicated FLAG-tagged EBP50 plasmids for 6 h and were immunostained with mouse anti-FLAG (M2) antibody (green) and Hoechst 33342 reagent (blue). Supplementary Figure 2. Down-regulated EBP50 expression does not affect β-catenin expression in SW480 cells. Two independent EBP50 knockdown SW480 stable clones were processed for double immunofluorescence study using EBP50 (red) and β-catenin (green) antibodies. Scale bars: 10 μm. Supplementary Figure 3. SW480 cells stably expressing FLAG-dnTCF-1 were transfected with EGFP-C1 control vector (C1) or EGFP-tagged EBP50 expressing plasmid (E50). Twenty four hours later, the cells were then processed for ChIP assay using affinity purified rabbit anti-FLAG, β-catenin antibody, or a rabbit anti-podocalyxin antibody as a negative control (Ctr). Target c-Myc and cyclin D1 sequences were amplified by PCR. Supplementary Figure 4. Mock (M) and EBP50 knockdown (Si) Colo205 cells were homogenized with Dounce homogenizer (20 strokes with a tight pestle) and centrifuged at 700 g for 5 min to separate cytosol and nuclei. Equal fractions from the cytosolic and nuclear portions were loaded into SDS-PAGE and analyzed by Western blotting analysis for EBP50, β-catenin, and TCF-1. Tubulin serves as a cytosolic marker while TCF-1 itself as a nuclear marker. * and ** indicate full-length and N-terminally truncated TCF-1, respectively. Supplementary Figure 5. (A) β-catenin immunostain displayed a similar increasing intensity and nuclear distribution pattern across the tumor to that seen for EBP50 staining shown in the analogous parts shown in Figure 8A. High magnification of areas *, **, and *** labelled in panel A showed β-catenin staining throughout the mucosal (B), submucosal (C), and deeper invasive portions (D) of the tumor. These regions are also correspondents of B, C, and D shown in Figure 8. (E) A CRC case showed membranous, cytosolic, but not any nuclear EBP50 staining at the invasive front despite the dysmorphic nuclear change. (F, G, H) High magnification of areas *, **, and *** labeled in panel E. Please note that there was still no EBP50 nuclear staining in the deeply invasive neoplastic glands in pericolonic soft tissue as shown in panel H. Scale bar, 50 μm for all panels; 10 μm for the insert in panel D. Supplementary table 1.