Figure 3
TGF-β and Wnt5a can inhibit the Wnt/β-catenin pathway. (a) Western blot of β-catenin protein from fractionated primary mammary epithelial cells after overnight stimulation with 60 ng/ml rWnt3a alone or in combination with 80 ng/ml rWnt5a. rWnt3a elevated nuclear β-catenin levels in epithelial cells, while rWnt5a inhibited rWnt3a-induced levels. (b) Immunofluorescent staining and confocal imaging for β-catenin in sections of WT and Wnt5a-/- mammary epithelium after three weeks of development illustrating nuclear and cytoplasmic localisation of β-catenin in Wnt5a-/- epithelium. A representative of five separate glands is shown. (c) Western blot shows the accumulation of β-catenin in the nuclear compartment of Wnt5a-/- epithelium relative to WT epithelium. (d) β-catenin localisation in MMTV-PyVmT and MMTV-PyVmT:Wnt5a-/- tumours (five tumours per group). The arrows point to examples of nuclei. (e) Subcellular fractionation and western blotting for β-catenin protein show elevated β-catenin levels in the nucleus of cells from Wnt5a-/- tumours. (f) Semi-quantitative RT-PCR using cDNA generated from individual MMTV-PyVmT and MMTV-PyVmT:Wnt5a-/- tumours, exhibiting increased Wnt/β-catenin transcriptional responses in the absence of Wnt5a (four control tumours, five Wnt5a-/- tumours). 18S was used to normalise for the amount of total RNA. PCR product is shown in the linear range (25 cycles for axin, tcf4, cyclin D1 and c-myc, and 15 cycles for 18S). (g) Immunofluorescent staining and confocal imaging (representative of four tumours per group), and (h) representative western blot of proteins from fractionated tumours, together, illustrating an increase in intracellular β-catenin when TGF-β signalling is blocked via overexpression of DNIIR. Arrows point to examples of nuclei. CREB is used as a loading control for the nuclear fraction, β-Tubulin is used as a loading control for the cytoplasmic fraction. DNIIR = dominant-negative TGF-β type II receptor; H&E = haematoxylin and eosin; MMTV = mouse mammary tumour virus promoter/enhancer; PyVmT = polyoma virus middle T antigen; RT-PCR = reverse transcripase polymerase chain reaction; TGF-β = transforming growth factor-beta; Wnt = Wingless-related protein family; WT = wild type, CREB+ c-AMO response element binding factor.