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Fig. 5. Transcriptionally active and inactive stabilized ß-catenin mutants inhibit neurite outgrowth. PC12 cells were transfected with GFP alone or one of three stabilized ß-catenin constructs shown on the left. Transcriptional activation (A) and NGF-induced neurite formation (B) and neurite growth (C) were measured. (A) Co-transfection of pTOPFLASH Tcf/Lef-driven luciferase reporter (gray bars, TOPFLASH) indicates that only full-length stabilized ß-catenin (*ß-catenin) increased Tcf/Lef-mediated transcription. pFOPFLASH (black bars, FOPFLASH) is a negative control for the reporter assay, with mutated Tcf/Lef-binding sites. Luciferase activity was normalized for transfection efficiency (see Materials and Methods). Shown is the mean±s.e.m. of three independent experiments. (B,C) One day after transfection, PC12 cells were passaged and treated with NGF for 3.5 days. Cells were stained for tubulin and >250 cells were counted for the presence of neurites (see Table 1). Expression of each stabilized ß-catenin mutant reduced the percentage of cells with neurites of any length (B) and cells with neurites longer than twice cell body width (C). For the GFP control, *ß-cat and ß-cat-eng bars show the mean±s.e.m. of three independent experiments. For *ß-cat
C-term, the bar represents the average of two independent experiments. Each mutant was significantly different from the GFP control by binomial test (*P<0.05). (D) Representative images of PC12 cells expressing CFP-tagged *ß-catC-term and induced with NGF for 24 hours. High-expressing cells (arrowheads) usually had short or no neurites in contrast to untransfected cells. (E) Quantification of neurite length in cells, including those in D, which express varying levels of *ß-catC-term and *ß-catenin. For each cell, higher mean fluorescence intensity correlated with decreased length of the longest neurite. Bar, 10 µm.
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