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Fig. 3. Exogenous Netrin-1 reverses Cripto-1-dependent epithelial-to-mesenchymal transition. (A) Western blot analysis of lysates from EpH4/Cr-1 cells cultured in growth medium containing 50 ng/ml of exogenous rmNetrin-1 (+NTN) showing increased expression of E-cadherin (E-cad), reduced expression of vimentin (Vim) and reduced expression of phosphorylated-Akt (P-Akt) compared to EpH4/Cr-1 cells cultured in growth medium alone (control). (B) Histogram summarizes results from invasion assays showing increased invasion of EpH4/Cr-1 and HC-11/Cr-1 cells as compared to wild-type cells. Addition of rmNetrin-1 (25 ng/ml and 50 ng/ml) significantly reduced invasion of both Cr-1 overexpressing cell lines. Values are the mean±s.d. of three separate experiments (*P<0.05 compared to levels in the relevant control group). (C) Western blot analysis of lysates from Cr-1 overexpressing cells shows that Neogenin expression decreases and UNC5H1 expression increases when these cells are treated with exogenous soluble rmNetrin-1 (+Netrin-1) as compared to untreated control cells. (D) Histogram summarizes results from invasion assays showing that the significant inhibition of invasion of EpH4/Cr-1 cells in the presence of 25 ng/ml of exogenous rmNetrin-1 is restored to control level when EpH4/Cr-1 cells are pretreated with neutralizing antibody against human UNC5C ({alpha}-UNC5) but not when pretreated with neutralizing antibody against Neogenin ({alpha}-NEO). Values are the mean±s.d. of three separate experiments (*P<0.05 compared to levels in the control group).