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Fig. 1. Stable expression of functional, full-length mycAPC in SW480 cells. (A) Expression of wild-type APC mRNA in SW480APC cells by RT-PCR analysis. SW480 cells were transfected with pEF-mycAPC or pEF and neomycin-resistant clones selected. Reverse-transcribed APC cDNA was amplified over the region containing the mutation in SW480 cells and digested with PstI, which cleaves only wild-type APC (Hargest and Williamson, 1995). LIM1215 colon cancer cells contain wild-type APC. RT-PCR products from untransfected SW480 cells and control SW480 clones (.2, .7 and .9) are not cleaved by PstI. SW480APC cells (pEF-mycAPC-transfected, independent clones .23, .15, .34, .24 and .38) contain both endogenous, mutated APC mRNA that cannot be digested by PstI (436 bp RT-PCR fragment) and APC mRNA that is cleaved by PstI (294 and 142 bp RT-PCR products), demonstrating the presence of ectopically expressed APC. Some SW480APC cells (clones .14, .18 and .5) that were transfected with pEF-mycAPC and selected in neomycin did not show expression of ectopically expressed APC by RT-PCR analysis. (B) Expression of APC results in translocation of ß-catenin to the cell periphery. SW480 (parental), SW480 control (pEF vector transfected, independent clones .2, .7 and .9) and SW480APC cells (pEF-mycAPC-transfected, independent clones .23, .15, .34, .24, .14, .18, .38 and .5) were stained immunochemically with antibodies to ß-catenin and Alexa488-conjugated anti-mouse IgG. Fluorescent staining was imaged by laser-scanning confocal microscopy. ß-catenin is predominantly nuclear in SW480 cells, SW480 control clones and SW480APC clones negative for APC by RT-PCR analysis, but translocates to the cell periphery in SW480APC clones containing ectopically expressed APC (.23, .15, .34, .24 and .38). Bar, 10 µm. The cellular distribution of ß-catenin was scored as nuclear and cytoplasmic (N), nuclear, cytoplasmic and peripheral (N/P), or peripheral and no nuclear (P), and graphed. Shown is a representative of at least two independent experiments; n, total number of cells counted. (C) ß-catenin/Tcf/LEF reporter activity is downregulated in SW480APC cells. SW480 and SW480APC (independent clones .15, .34 and .24) cells were transfected in triplicate with Tcf/LEF luciferase (TOPflash) reporter gene constructs (van de Wetering et al., 1997). Shown is the ratio of TOPflash to FOPflash luciferase activity from a representative of at least three independent experiments. Error bars indicate s.e.m. from triplicate measurements. (D) Western blot analysis demonstrating expression of full-length mycAPC in SW480APC cells. SW480 and SW480APC cells were lysed, proteins separated by 4% SDS-PAGE and immunoblotted using an anti-myc (9E10) mAb. (E) Western blot analysis of total cellular levels of ß-catenin in SW480APC cells. SW480, SW480 control and SW480APC cells were lysed, 5 µg protein separated by 4-20% SDS-PAGE and immunoblotted using an anti-ß-catenin mAb and an anti-actin mAb to control for protein loading (lower panel). The level of ß-catenin in each clone was quantitated by densitometry, normalized to actin, and is represented as a ratio to ß-catenin in SW480 cells. Shown is a representative of at least three independent experiments.
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