Figure 11

Minimal TCF reporter activity and lack of nuclear accumulation of mutant β-catenin in poorly differentiated SNU449.cl8 cells. SNU449 cells were stably transfected with Tet-responsive ΔN-β-catenin expression vector to obtain SNU448.cl8 cells. (a) Induced expression of N-terminally truncated ΔN-β-catenin protein in the Tet-Off conditions, as tested by western blot assay. Total cell lysates were extracted from cells and subjected to western blot assay using anti-β-catenin antibody. (b) TCF activity is only weakly induced in Tet-off conditions, as tested by duplicate experiments. (c) SNU449.cl8 cells at Tet-On state express wild-type endogenous β-catenin protein principally located at cell membrane. Under Tet-Off conditions the staining pattern remains almost identical despite ΔN-β-catenin expression. Note lack of nuclear accumulation. SW480 cells used as positive control display strong nuclear β-catenin staining. Cells were grown on coverslips, subjected to indirect immunofluorescence assay using anti-β-catenin antibody (red), counterstained with DAPI (blue) and examined by confocal microscopy.