Figure 3: Wnt secretion is required to maintain canonical Wnt signalling in colon cancer cell lines.

(a) DLD1 and SW480 colon cancer cells activate the Wnt pathway in cocultured MDA-MB231 reporter cells. DLD1 or SW480 cells were cocultured with MDA-MB231 reporter cells stably transduced with a TCF/LEF reporter (1,500 cells) at the indicated ratio for 48 h. Next, cells were lysed and luciferase activity was measured. (b) Silencing of Wnt3 in DLD1 and HCT116 cells stably transfected with a TCF4/Wnt reporter reduces canonical Wnt signalling activity. Both cell lines were transfected with the indicated siRNAs. HCT116 cells were incubated for 72 h and DLD1 cells for 96 h before read-out. Luciferase levels were normalized to cell survival assessed by a CellTiterGlo assay performed in parallel. (c) Silencing of Wnt3a or Wnt3 reduces Axin2 protein expression. HCT116 cells were transfected with the indicated siRNAs for 72 h. Cell lysates were used for western blotting with indicated antibodies. β-Actin served as the loading control. (d) Evi/Wls knockdown in DLD1 cells reduces the induction of TCF/LEF reporter activity in coseeded MDA-MB231 reporter cells. Coculture of cells was conducted as described in a. Half times more DLD shEvi cells than shCtrl cells were seeded to compensate for reduced viability. (a,b,d) Data from three independent experiments are presented as mean±s.e.m.