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. 2008 Sep 4;27(19):2603–2615. doi: 10.1038/emboj.2008.178

Figure 1.

Figure 1

E-cadherin loss in epithelial cell lines induces either a cadherin switch or full EMT. (A) NMuMG, MCF7 and MTflEcad cells adapt a mesenchymal phenotype upon loss of E-cadherin by TGFβ treatment (+TGFβ), shRNA expression (shEcad) or Cre-mediated deletion of the E-cadherin gene (+Cre). (B) Immunoblot analysis for E-cadherin (Ecad), N-cadherin (Ncad), β-catenin, vimentin and NCAM expression in MCF7 and MTflEcad cells expressing E-cadherin (shCont and MTflEcad) or depleted for E-cadherin expression by shRNA expression (shEcad) or Cre-mediated deletion of the E-cadherin locus (MTdeltaEcad). Cell extracts were loaded on different gels and resolved proteins were visualized with specific antibodies as indicated. Immunoblotting for vinculin was used as a loading control. (C) NCAM expression is induced upon loss of E-cadherin function. NCAM mRNA levels were determined by quantitative RT–PCR in MCF7, MTflEcad and NMuMG cells that have been depleted for E-cadherin (−E-cadherin) or not (+E-cadherin). For all values P<0.0001, unpaired t-test. Data are shown as mean±s.d. *As NCAM is not expressed at detectable levels in MCF7 cells, its upregulated expression in MCF7-shEcad cells cannot be calculated as fold induction. Instead the comparative Ct values (ΔΔCt) are given in the lower row. (D) Immunoblot analysis for NCAM protein expression in NMuMG cells in the presence of TGFβ at the indicated time points. Immunoblotting for vinculin was used as a loading control. (E) NCAM mRNA levels were determined by quantitative RT–PCR in NMuMG cells treated with TGFβ for the times indicated. *P=0.0134, **P<0.005; unpaired t-test). Data are shown as mean±s.d. (F) NCAM promoter activity is increased upon loss of E-cadherin expression. NMuMG were transfected with a CAT reporter plasmid containing 1000 bp (ΔSsp) or 647 bp (ΔNde) of the NCAM promoter sequence and treated with or out TGFβ for 6 days prior to the analysis of CAT activity (left panel; *P=0.0027 and **P=0.0007, unpaired t-test). Data are shown as mean±s.d. (G) NMuMG cells stably transfected with shControl or shSmad4 were treated for 3 days with TGFβ and then analysed for NCAM, E-cadherin (Ecad) and N-cadherin (Ncad) expression by immunoblotting analysis. Immunoblotting for vinculin was used as a loading control.