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Fig. 1. Generation of HCT-116 cells stably expressing N-terminal APC fragments. (A) Schematic of the APC protein (Mimori-Kiyosue and Tsukita, 2001) and the three N-terminal fragments used to create the stable cell lines, showing the binding sites for Asef (blue), ß-catenin/GSK3ß (pink, green), axin (orange), microtubules (purple), EB1 (yellow) and hDLG (black). (B) Protein extracts from tetracycline inducible 293 cells stably expressing myc-tagged N750 (lane 1), N1309 (lane 2), N1807 (lane 3) and full length APC (lane 4), blotted to detect myc-tagged proteins (left) and APC (right). The asterisk indicates a background band, while the arrow indicates endogenous full length APC. (C) Cloning efficiency of the cell lines indicated in either 10 µg/ml Zeocin^TM (upper panel) or 200 µg/ml hygromycin B (middle panel). ß-Galactosidase activity exhibited by the cell lines is shown in the lower panel. (D) HCT-116-derived cells stably transfected with Myc-tagged control or N-APC vectors were stained with anti-Myc antibodies (red) and Hoechst dye (blue) to visualise the DNA. Only background staining is observed in the control line, whereas nuclear/cytoplasmic staining is visible in the N-APC lines. (E) Protein extracts from HCT-116 control (lane 1) and N750 cells (lane 2), and 293 N750 cells, uninduced (lane 3) or induced with tetracycline (lane 4) blotted to detect endogenous full length APC (upper panel) and Myc-tagged proteins (lower panel). The asterisk indicates a background band in the HCT-116 samples detected by the anti-myc antibody.

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