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Fig. 1. E-cadherin represses fibronectin and LEF1 gene expression. (A) Levels of E-cadherin and Snail1 proteins in SW-480 transfectants. SDS-protein extracts obtained from SW-480 cells stably transfected with the indicated genes and grown until 50-60% confluence were analysed by western blot (WB) with the indicated antibodies. (B) E-cadherin expression downregulates fibronectin and LEF1 RNA levels. Fibronectin and LEF1 RNAs were determined by qRT-PCR in SW-480 cell lines. Values are relative to that obtained in control SW-480 cells. Graphics show the average ± s.d. of the three values obtained for every sample. (C) E-cadherin expression downregulates fibronectin and LEF1 promoter activity. Activities of –341/+265 fibronectin promoter and –735/+1077 LEF1 promoter were determined after transfection of these promoters, which were inserted into pGL3 plasmid as described, into subconfluent SW-480 stable transfectants. The values show the average ± s.d. of two experiments performed in triplicate samples, and are relative to the value obtained in control SW-480 cells. (D) Cell-culture confluence regulates SW-480 E-cadherin levels. SW480 ADH cells were grown in standard conditions until 50-60% confluence (Sub-Conf) or 3 days after 100% confluence (Conf). 1% SDS total protein extracts were obtained and analysed by western blot with anti-E-cadherin or anti-pyruvate-kinase mAbs. (E) Expression of fibronectin and LEF1 decrease in confluent SW-480 cells. Fibronectin and LEF1 RNAs were determined by qRT-PCR and values (average ± s.d.) referred to the value obtained in the sub-confluent cells. (F,G) Interference of E-cadherin expression upregulates fibronectin and LEF1 RNA levels. Cells expressing an siRNA specific to E-cadherin or a scrambled control (Irr) were cultured until confluence, and E-cadherin and actin levels were determined by western blot (F). In parallel, fibronectin, LEF1 or HPRT RNA content were determined in these cells by semi-quantitative RT-PCR (G).
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