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First published online March 18, 2009
doi: 10.1242/10.1242/jcs.028241

Journal of Cell Science 122, 1014-1024 (2009)
Published by The Company of Biologists 2009
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The class I bHLH factors E2-2A and E2-2B regulate EMT

Verónica R. Sobrado1, Gema Moreno-Bueno1, Eva Cubillo1, Liam J. Holt1,*, M. Angela Nieto2, Francisco Portillo1 and Amparo Cano1,{ddagger}

1 Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Madrid (UAM), Instituto de Investigaciones Biomédicas `Alberto Sols' (CSIC-UAM), 28029 Madrid, Spain
2 Instituto de Neurociencias de Alicante, CSIC-UMH, 03550 Sant Joan d'Alacant, Spain


Figure 1
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  		Fig. 1. E2-2B induces a full EMT when overexpressed in MDCK cells. (A) Phenotypic characterisation of MDCK-E2-2B cells. Phase-contrast images of MDCK-CMV control cells (a) and one representative clone from MDCK-E2-2B cells, C2 (b). Scale bar: 100 µm. Immunofluorescence images of EMT markers, in MDCK-CMV (c,e,g,i) and MDCK-E2-2B, C2 cells (d,f,h,j). E-cad, E-cadherin; FN, fibronectin; N-cad, N-cadherin; VM, vimentin. F-actin stain in MDCK-CMV (k) and MDCK-E2-2B (l) cells. White arrows indicate lamellipodia-like structures. Scale bar: 50 µm. (B) Western blot analysis of E2-2B-HA and the indicated epithelial and mesenchymal markers in MDCK-CMV and three independent clones from MDCK-E2-2B cells. {alpha}-Tubulin levels are shown as a loading control. (C) Immunofluorescence images showing the nuclear localization of E2-2B-HA protein. Panels a,b: control MDCK-CMV; c,d: MDCK-E2-2B, C2 cells. Scale bar: 40 µm. (D,E) Semiquantitative RT-PCR analysis to detect the expression of the exogenous E2-2B (D, upper panel) and endogenous Cdh1 (D, middle panel) transcripts and of endogenous dog E47, Snail1and Snail2 in the indicated clones and control cells (E). GAPDH levels are shown as a loading control.

 

Figure 2
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  		Fig. 2. E2-2B represses E-cadherin gene promoter activity in MDCK cells. (A) The activity (mean ± s.d.) of mouse proximal wild-type Cdh1 promoter (–178/+92) fused to the luciferase gene analyzed in MDCK cells in the presence of increasing amounts (25-200 ng) of pcDNA3-E2-2B-HA or empty pcDNA3-HA control vector (200 ng). (B) The activity of the wild type (WT) Cdh1 promoter (–178/+92) and versions containing point mutations in E-box1 (MutE1), E-box2 (MutE2), E-box3 (MutE3) or in both E-box1 and E-box2 (MutEpal) (see scheme) were analyzed in MDCK cells in the presence of pcDNA3-E2-2B-HA or empty pcDNA3-HA as indicated. CMV-β-gal was used to normalize transfection efficiency. Luciferase and β-galactosidase activities were determined 24 hours after transfection. The promoter activity is represented as relative luciferase units (RLU) and the values were normalized to those obtained in the presence of control vector. A representative experiment out of at least three experiments is shown in each panel, each performed in triplicate. Error bars represent s.d. (C) Chromatin immunoprecipitation assays performed with an anti-HA antibody in MDCK-CMV-HA, MDCK-E2-2B-HA (clones C1, C3) and MDCK-Snail1-HA cells. Amplified fragment corresponds to position –85/+130 of the dog Cdh1 gene. Controls obtained in the absence of antibody (–) and after precipitation with an irrelevant antibody (IgG), as well as inputs, are included.

 

Figure 3
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  		Fig. 3. E2-2B overexpression causes the acquisition of a migratory and invasive behaviour. (A) The migratory capability of MDCK-E2-2A (C1,C3,C8), MDCK-E2-2B (C1,C2,C3), MDCK-Snail1 and MDCK-CMV control cells was analyzed in Transwell assays on type IV collagen. Results represent the mean ± s.d. of three experiments performed in duplicate samples. *P<0.05; **P<0.01; ***P<0.001 in Student's t-test with respect to MDCK-CMV cells. (B) The infiltrating behaviour of MDCK-CMV (a-d) and MDCK-E2-2B, C2 (e-h) cells was analyzed in three-dimensional organotypic cultures on type I collagen gels. Immunofluorescence images of E-cadherin (red; a,e) and vimentin (green; b,f) from 8 µm cryostat sections from 2 week cultures. Nuclei staining with DAPI corresponding to the same fields of MDCK-CMV (c,d) and MDCK-E2-2B (g,h) cultures are also shown. Scale bar: 100 µm.

 

Figure 4
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  		Fig. 4. Comparative gene expression analysis between MDCK-E2-2 and MDCK-E47 cells. (A) Unsupervised cluster analysis of genes modified in MDCK cells expressing E2-2 (blue) or E47 (yellow) factors, when compared with control MDCK-CMV cells. Only genes with twofold or greater variation in at least one cell type were considered and three hybridizations of each cell type were analyzed. Upregulated (red) or downregulated (green) genes are indicated. (B) Graphic representation of the common and specifically regulated genes in MDCK cells expressing E2-2 and E47. A total of 177 genes were differentially expressed more than twofold in at least one cell type relative to control cells. The number of genes found to be regulated in each case is indicated inside each bar; upregulated (red) or downregulated (green) genes are indicated. (C) Functional grouping of the modified expressed genes. The specific functions are listed on the right using a colour code and numbers indicate the percentage representation among all the candidate genes. (D) Validation of selected genes. Left, RT-PCR analysis of endogenous SPARC, ZEB1 and PDGF-C transcripts in the indicated cell lines. Right, western blot analysis of SPARC and Id1 protein levels in the indicated cell lines. {alpha}-tubulin (right) and GAPDH (left) are shown as loading controls. Two independent clones from MDCK-E2-2B and MDCK-E2-2A cells were analyzed. MDCK-E47 cells were also included.

 

Figure 5
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  		Fig. 5. Tcf4 is expressed in the mesoderm but not in embryonic epithelia. In situ hybridization of whole-mount mouse embryos at 8.5 d.p.c. (A-B) and 10 d.p.c. (C-D), and transverse vibratome sections of 10 d.p.c. embryos taken at the level of the anterior (E-F) and posterior trunk (G-H). Tcf4 products are detected in many different tissues throughout the embryo including the mesoderm, limb buds and neural tissues but are absent from the non-neural epithelia (E,G), the heart primordium (A) and the extra-embryonic membranes (A,G). E2A (E47) transcripts show a similar expression pattern, but with a wider distribution in the mesoderm and increased levels in the neural tube (F,H), compared with Tcf4 transcripts (E,G). Note that the region of the neural tube undergoing EMT (EMT zone) expresses very low levels of Tcf4 and E2A (E47) (E,F). a, amnion; ba, branchial arch; ect, ectoderm; end, endoderm; em, extraembryonic membranes; h, heart; lb, limb bud; mnc, migratory neural crest cells; nt, neural tube; s, somites.

 

Figure 6
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  		Fig. 6. Interference of Tcf4 by shRNA. MDCK cells stably expressing E2-2B (A,B), E47 (C,D) or Snail1 (E,F) factors were transfected with shE2-2.1 or control vector and selected as pooled GFP-positive cells. Cells were analyzed by RT-PCR for expression of the indicated exogenous transgenes (A,C,D) and endogenous transcripts (A-F). Analysis of endogenous transcripts in untreated MDCK-CMV cells (B,D,F) was included as an additional control. Expression of E2-2B transgene was also confirmed by western blot analysis in MDCK-E2-2B cells (A, lower panels). (G) The expression of E-cadherin in MDCK-CMV control and indicated pooled cells after transfection with shControl (C) or shE2-2 (RNAi) was analyzed by RT-PCR (upper) and western blot (lower). GAPDH and {alpha}-tubulin levels were detected to control cDNA and protein loading, respectively.

 

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