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Fig. 6. Altered ß-catenin subcellular distribution and effect of viral transduction of constitutively active ß-catenin in OG2-NcadC cells. (A) The cytosolic/membrane and nuclear fractions of calvaria cells (CLVC) isolated from newborn OG2-NcadC (Tg) or wild-type (WT) mice (left panel) or of bone marrow stromal cells (BMSC) isolated from 4-month-old mice (right panel) were separated as described in Materials and Methods, and blotted using an antibody that recognizes the cytoplasmic tail of N-cadherin. Note that OG2-NcadC-expressing cells exhibited more abundant ß-catenin in cytosol/membrane compartments and lower ß-catenin abundance in the nuclear fraction, relative to wild-type cells. The distribution of ß-catenin between cytosol/membrane and nuclear compartments is similar in transgenic and wild-type cells. (B) OG2-NcadC and wild-type CLVC were transduced with retroviral vectors expressing either the stable, constitutively active ß-catenin construct, N151 or LacZ, and cultured for 2 weeks before determination of alkaline phosphatase activity. Transduction of ß-catenin increased enzyme activity in OG2-NcadC CLVC to levels similar to those present in wild-type cells, whereas no effect was seen in either nontransduced cells or cells exposed to LacZ retrovirus. (C) OG2-NcadC and wild-type BMSC were transduced with the N151 or LacZ retroviral vectors, as just described, and grown for 2 weeks for CFU-Adipocyte determination. Transduction of ß-catenin reduced the number of CFU-Adipocyte in OG2-NcadC BMSC to levels similar to those present in wild type cells, while transduction of LacZ was ineffective; *P<0.05, t-test (mean±s.e.m.). All data are representative or the average of at least three different experiments.

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