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Figure 4


Fig. 4. E-cadherin inhibits NF-{kappa}B transcriptional activity on the fibronectin promoter. (A) Binding of NF-{kappa}B to the fibronectin promoter is sensitive to E-cadherin. ChIP assays were carried out as described in the Materials and Methods, immunoprecipitating crosslinked nuclear extracts from SW-480 cells stably transfected with the indicated genes. Semi-quantitative analysis from one experiment of the three performed (right) or the average ± s.d. of quantitative analysis of three experiments (left) is shown. (B) E-cadherin controls p65 association to DNA. Gel shift assays were performed as described in the Materials and Methods, with an oligonucleotide containing the NF-{kappa}B-binding element present in the human fibronectin promoter. Nuclear extracts form SW-480 cells transfected with Snail1 alone or both Snail1 and E-cadherin were used. In the experiment shown in the left panel, binding of the radioactive probe was competed with a 50- or 100-fold excess of non-radioactive probe containing a consensus binding element for NF-{kappa}B, either wild-type (WT) or mutated (MUT). When indicated (right panel), binding was carried out in the presence of an irrelevant IgG or a mAb specific for p65, as indicated in the Materials and Methods. The arrows show the specific band detected with this assay; the arrowhead shows the migration of the free probe. Note that the upper band was not competed either by the NF-{kappa}B consensus oligonucleotide or by the p65 antibody; therefore, we considered that it did not correspond to a p65 complex. The results of a representative experiment of the four (right) or five (left) that were performed are shown. (C) Snail1 and E-cadherin modulate NF-{kappa}B transcriptional activity. The activity of an NF-{kappa}B-dependent promoter (NF3) was determined in SW-480 cells stably transfected with Snail1-HA, E-cadherin or both. The same experiment was performed in MiaPaca-2 cells transiently transfected with these two cDNAs. The results correspond to the average ± s.d. of three experiments.





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