Figure 2

APC knockdown in 4T07 cells perturbs β-catenin localization at protrusion ends but does not induce β-catenin/TCF-transcriptional activity. (A) Apc mRNA expression was analyzed in 4T07 stable pools expressing vector only (pLKO.1), a scrambled (scr) shRNA and two independent APC-specific shRNAs by qRT-PCR. Values were normalized to 18S rRNA expression, and data are presented relative to expression in pLKO.1 control cells, set at 100% (*p < 0.05 compared to pLKO.1). (B) Lysates from 4T07 control and APC-knockdown cells were subjected to western blotting with an anti-APC antibody [19]. Lysates from HCT116 cells are included as a control for full-length APC. Densitometry values are APC levels normalized to actin relative to the pLKO.1 control cells. (C) IF with anti-APC and -β-catenin antibodies illustrate co-localization to membrane protrusion ends. In contrast, APC-knockdown cells lack specific localization of APC or β-catenin to protrusion ends and generally have a more rounded morphology. Images were taken at the same exposure level for APC protein; cytosolic staining is non-specific. 630X. (D) Cells were transfected with pTOPFlash containing TCF consensus sites upstream of luciferase or pFOPflash with mutant TCF-binding sites. Transfection efficiency was normalized by transfection of pRL-TK, and the data are plotted as the TOPflash/FOPflash ratio. SW480 cells are a positive control, and there are no significant differences between APC-knockdown and control 4T07 cells. (E) BrdU incorporation assays were performed on APC-knockdown and control cells, and the percentage of cells with BrdU incorporation after 24 h was quantified by IF with an anti-BrdU antibody and plotted.