Figure 1.
Id1 is upregulated and interacts in vivo with E47 in MDCK-EGFP-E47 cells.
(A) Cell extracts derived from MDCK-EGFP and MDCK-EGFP-E47 cells were analysed for expression of EGFP-E47, Id1 and Id3 proteins by Western blot; alpha-tubulin was used as loading control. Two independent clones (#2, #6) generated after stable expression of EGFP-E47 in MDCK cells are shown. (B) Cell extracts derived from MDCK-EGFP and MDCK-EGFP-E47 cells were immunoprecipitated with anti-Id1 antibodies and control IgGs and analysed by Western blotting with the indicated anti-Id1 and anti-E2A antibodies. (C) Nomarski (a,b) and confocal immunofluorescence images (c-h) of MDCK-EGFP and MDCK-EGFP-E47 cells. Localization of EGFP (c,g), EGFP-E47 (d,h) and Id1 (e,f) is shown. See the colocalization of Id1 with EGFP-E47 in the nuclei (merged image, h). Bars, 25 um. (D) Cell extracts derived from MDA-MB435S (left panels) and A375P (right panels) cells were immunoprecipitated with anti-Id1 antibodies and control IgGs and analysed by Western blotting with the indicated anti-Id1 and anti-E2A antibodies.
Figure 2.
E47 represses E-cadherin by direct binding to the endogenous promoter in complexes devoid of Id1.
(A) Dependence of E47 repression of E-cadherin promoter on proximal E-boxes. HEK293T cells transiently co-transfected with 200 ng of the proximal wild type mouse E-cadherin-Luciferase promoter (−178/+92 bp) or point mutants in the proximal E-boxes (E-pal +/− E-box3) and 10 ng of CMV-beta-gal plasmid in the presence of 50 ng of pcDNA3-E47 (dark grey bars) or control pcDNA3 (light grey bars) vectors, as indicated. Differences between the E47 mediated repression of the mutants versus the wild type promoter were statistically analysed by t-Student test (***p<0.001). (B) and (C) E-cadherin promoter activity in MDCK (B) and MDCK-CMV and MDCK-E47 (C) cells transiently co-transfected with 200 ng of the proximal mouse E-cadherin-Luciferase promoter (−178/+92 bp) and 20 ng of TK-Renilla (B) or 10 ng of CMV-beta-gal (C) plasmids, in combination with the indicated amounts of pcDNA3-E47 and/or pcDNA3-Id1 vectors. RLU is normalized to that detected with empty pcDNA3 vector (A, B) or in control MDCK-CMV cells (C, left); results represent the mean of at least two independent experiments +/− s.d. (D) and (E) Chromatin immunoprecipitation (ChIP) assays of EGFP-E47 at the endogenous E-cadherin promoter in MDCK-EGFP-E47 cells. (D) ChIP assays were first performed with an antibody specific for EGFP or control IgG and the bound fraction analysed for endogenous E-cadherin promoter sequences (dCdh1) (left three lanes), followed by re-ChIP with anti-Id1 antibodies (IP2) and analyses of the bound (B) and unbound (NB) fractions for E-cadherin promoter sequences (dCdh1) (right two lanes). The input fraction (1/100) is shown at the left. Quantification of the amplified endogenous E-cadherin promoter compared to the input fraction (1/100) is shown in the lower panel. (E) ChIP assays were first performed with anti-EGFP (IP1) followed by re-ChIP (IP2) with anti-EGFP or anti-Id1 antibodies, as indicated, followed by analyses of the bound (B) and unbound (NB) fractions for E-cadherin promoter sequences (dCdh1). Input (1/100) and control IgG ChIP are shown on the left two lanes. Quantification of the amplified endogenous E-cadherin promoter compared to the input fraction (1/100) is shown in the lower panel.
Figure 3.
E47-silencing in MDCK-EGFP-E47 cells induces a MET process, downregulation of Id1 and reduction of tumour growth.
(A) Phase contrast images (a-e) and immunofluorescence images (f–y) of the indicated cell lines showing localization of EGFP (f,g), EGFP-E47 (h–j), Ecadherin (k–o), beta-catenin (p–t) and F-actin organization (u–y). Nuclei were stained with DAPI (k–y). Black bar, 60 um (a–e); white bars, 25 um (f–y). (B) Western blot analyses of epithelial markers and Id1 in the indicated cell lines; alpha-tubulin was used as loading control. Two (#N, #P) and five independent clones (#N, #M, #L, #O, #P) generated after stable expression of shEGFP into MDCK-EGFP-E47 (clone #6) cells are shown in A and B, respectively. (C) The tumorigenic potential of MDCK-EGFP-E47#6 and MDCK-EGFP-E47-shEGFP#N and #P cells was analysed by subcutaneous injection into nude mice. ANOVA analysis: *p<0.05; **p<0.01; ***p<0.001. (D) Histology (upper panels) and immunohistochemical analysis for E-cadherin (lower panels) of tumours induced by MDCK-EGFP-E47#6 (left) and MDCK-EGFP-E47-shEGFP#N cells (right). Amplification x20. Insets show amplified fields at x40.
Figure 4.
Id1 knockdown in MDCK-EGFP-E47 cells induces a partial MET and impairs cell survival.
(A) Western blot analysis of Id1 expression in MDCK-EGFP-E47 cells after 48 h infection with shId1 or control lentivirus (EV), compared to non-infected (EGFP-E47) and control MDCK-CMV (CMV) cells; alpha-tubulin was used as loading control. (B) Phase contrast (upper panels) and confocal immunofluorescence images of E-cadherin (lower panels) of EGFP-E47-shId1 (right) and EGFP-E47-EV control (middle) cells 48 h after lentiviral infection; non infected MDCK-EGFP-E47 cells (left) are shown as additional control. Nuclei were stained with DAPI; bars, 50 um. (C) Cell cycle analysis of the indicated cell lines by propidium iodide staining and FACS analysis. (D) Apoptosis rate of the indicated cells determined by the sub-G0 population. Results in C and D represent the mean +/− s.d. on triplicate samples.
Figure 5.
E47 and ID factors are preferentially expressed in basal-like breast carcinomas.
(A) Unsupervised hierarchical clustering of the vant’Veer dataset (ref. 36) for the expression of the indicated basal and luminal markers, together with estrogen (ESR1) and progesterone receptors (PGR) and ErbB2, identifies two clusters of basal (yellow) and non-basal (purple) tumours. Increased TCF3 and ID1/ID4 transcripts cluster with the basal-like subgroup. (B) Association between the expression of TCF3 and ID1/ID4 with the basal-like phenotype in the vańt Veer's dataset. To validate the association among specific genes and/or tumour subgroups, each variable was categorized using the percentile 75 value as the cut-off. (C) Overexpression of TCF3 associated to basal-like breast tumours (upper) and to decreased overall survival (bottom) in the indicated public datasets.