Figure 2

TamR cells exhibit increased Wnt signalling. a: mRNA expression of Wnt-related genes was measured using qPCR. Graph represents the mRNA fold-regulation values of TamR cells relative to MCF7 cells, normalized against three housekeeping genes with standard deviation (s.d) of triplicate experiments represented by error bars. *P<0.05, ** P<0.01. b: mRNA expression changes of Wnt target genes were measured using RT Profiler PCR arrays and normalised to five housekeeping genes. Criteria for significant change include a statistically significant validation determined by the manufacturer and fold regulation of greater or less than 4. c: MCF7 and TamR cells were co-transfected with pRL-TK (Renilla) and either TOPflash or FOPflash expression plasmids. Cells were subsequently treated with 0.1 μg/ml recombinant Wnt3a (rWnt3a) for 24 hours prior to luciferase activities being measured using a Glomax 96 Microplate Luminometer. Average activity and s.ds were derived from octopulate transfected samples. Results represent the average of 3 experiments and bars represent the s.d of the mean. ***P<0.001. d: MTT proliferation assays were performed on parental MCF7 cells and TamR cells treated with 5 μM Tamoxifen and 0.1 μg/ml recombinant Wnt3a (rWnt3a) for 24 hours. 50% DMSO was used as a control. Treatment with rWnt3a and 5 μM Tamoxifen inhibited the proliferation of both the MCF7 and TamR cells compared to treatment with Tamoxifen alone. rWnt3a treatment in the TamR cells enhanced their resistance to Tamoxifen back to near basal levels. Graph represents the average cell proliferation in percentage of 10 replicates with s.d represented by error bars. *P<0.05, *** P<0.001. e: mRNA expression of EMT markers was measured using qPCR. Graph represents the mRNA fold-regulation values of TamR cells relative to MCF7 cells, normalized against three housekeeping genes with s.d of five independent experiments represented by error bars. **P<0.01, ***P<0.001*.