The results of our study are relevant for the clinic, as VDR expression is downregulated in approximately AbMole Indinavir sulfate two-thirds of advanced colon tumors associated to the upregulation of SNAI1 and SNAI2 genes that code for SNAIL1/SNAIL2 transcriptional repressors. VDR loss may contribute to overactivate the Wnt/b-catenin pathway and so, to accelerate tumor growth and malignization. A few studies have proposed VDR expression as a marker of good prognostic in colon cancer and showed its correlation with high tumor differentiation and absence of node involvement. However, the low number of patients analyzed does not allow definitive conclusions. Patients with carcinomas at early stages that still express VDR could benefit from therapy with 1,252D3 compounds that would potentially reduce Wnt/b-catenin oncogenic function. However, when VDR expression is lost at advanced stages, tumors will not only be refractory to this type of treatment but will also acquire higher levels of nuclear b-catenin and enhanced Wnt/b-catenin signaling. For tissue immunofluorescence, formalin-fixed paraffin-embedded sections of mouse small intestine and colon or human colon tumors were prepared and immunolabelled as described elsewhere. Briefly, tissues were sectioned at 4 mm, deparaffined and rehydrated using xylene and a series of graded ethanol. After four washes in PBS, cells were incubated with secondary fluorophore-conjugated AbMole Diosgenin-glucoside antibodies and Hoechst 33342 for 1 h at RT, washed and mounted in Vectashield. Cell imaging was performed as described above for cell immunofluorescence. In brief, we defined Regions of Interest and obtained the integrated density of the ROI for the green and the red channels corresponding to specific antibody staining. In parallel, we measured the Hoechst integrated density of each ROI and divided it by a standard nucleus signal. The standard nucleus signal was calculated as the average integrated density of ten nuclei taken at random. The resulting values corresponded to the number of nuclei and, therefore, to the number of cells present in each ROI. We finally divided the integrated density of the proteins of interest in the ROI by the number of cells present in the same ROI. This value indicates the level of expression of the proteins of interest per cell. To measure nuclear b-catenin levels we previously used MacBiophotonics ImageJ software to generate an image where the positive staining corresponded to coincident Hoechst and b-catenin signals. To quantify the level of nuclear b-catenin in SW620 cells ectopically expressing different VDR variants, we adapted the above mentioned method to single cell measurements. Nuclear b-catenin level was calculated in cells transfected with empty vector or with wild type or mutant VDR. One hundred cells expressing exogenous VDRs or the empty vector were estimated in each condition and values were represented as arbitrary units.