Figure 2: Evi/Wls is required to maintain canonical Wnt signalling in colon cancer cell lines.

(a) Evi/Wls depletion reduces canonical Wnt signalling. DLD1 and HCT116 TCF4/Wnt reporter-colon cancer cells were transduced with lentivirus introducing a control or Evi shRNA construct. Luciferase assay was normalized to viability assessed in parallel using the CellTiterGlo assay. Results of three (for DLD1) and five (for HCT116) independent experiments are shown as mean±s.e.m. (b) Silencing of Evi/Wls regulates the Wnt target gene expression. DLD1 and HCT116 cells were transduced with a dox-inducible shmirEvi#1 construct and treated with or without dox. After 96 h, the cells were analysed for the expression of AXIN2 and EVI/WLS mRNAs by quantitative PCR. Results of four independent experiments are shown as mean±s.e.m. (c) Silencing of Evi/Wls reduces the level of active β-catenin in DLD1 and HCT116 cells, which express mutated APC or β-catenin, respectively. DLD1 cells were transfected with the indicated siRNAs for 120 h, and HCT116 cells were transfected for 72 h. Subsequently, the cells were lysed and western blotting was performed with the indicated antibodies. β-Actin served as the loading control. The reduction of total Lrp6 in β-catenin knockdown cells, which is consistently observed in DLD1 cells, may be a consequence of reduced β-catenin levels at the adherent junctions⁵⁹.