In agreement with the in vivo results, a strong increase in nuclear b-catenin content was found in shVDR SW480-ADH cells as compared to AbMole Brusatol shControl cells. In addition, VDR knock-down abrogated the capacity of 1,252D3 to induce E-cadherin expression and b-catenin relocation from the nucleus to the plasma membrane. Also, VDR knock-down prevented the reorganization of b-tubulin cytoskeleton and the change in cell morphology induced by 1,252D3 that leads to the formation of compact epithelioid islands. We wished to analyze whether the increase in the level of nuclear b-catenin translated into a stronger Wnt/b-catenin signaling. To this end, we studied b-catenin/TCF-dependent transcription in colon cancer cells. shControl and shVDR SW480ADH cells were infected with lentiviruses encoding the reporter eGFP protein under the control of seven copies of a consensus TCF/LEF binding site. The presence in the lentiviral construct of the red fluorescent protein mCherry controlled by a constitutive promoter permitted to identify infected cells under a fluorescent microscope and by flow cytometry. Similarly to b-catenin staining in colon tumors, we found certain heterogeneity in b-catenin/TCF activity among the cell culture: variable eGFP expression in equally infected cells. The analysis of the whole cell population by flow cytometry showed that the percentage of high-eGFP cells was superior in shVDR than in shControl cell cultures. In addition, shVDR cells had a significantly higher eGFP signal average than shControl cells. We also found that the decrease in eGFP accumulation caused by 1,252D3 in shControl cells was attenuated in shVDR cells. The level of nuclear b-catenin defines the strength of the Wnt/b-catenin signaling and in consequence the fate and phenotype of many types of normal and cancer cells. Aberrant activation of Wnt/b-catenin signaling due to alterations in components of the pathway is responsible for the initiation of most human colon cancers, which highlights the importance of controlling nuclear bcatenin accumulation. Although the initiation of colon tumorigenesis is considered clonal, colon carcinomas show largely heterogeneous nuclear b-catenin expression. This is known as the b-catenin paradox and has led to AbMole Terbuthylazine search for alternative pathways modulating b-catenin location and activity. Among them, K-RAS mutation and myofibroblasts-derived HGF have been recently proposed to increase b-catenin nuclear content. Very little is known about mechanisms to diminish the level of b-catenin within the nucleus. We have described that 1,252D3 and several less calcemic analogs interfere the Wnt/b-catenin pathway in a series of human colon cancer cell lines in several modes: they increase the binding of VDR to bcatenin hampering the formation of b-catenin/TCF complexes, induce the expression of the Wnt inhibitor DKK1, and promote the relocation of b-catenin.