Fig. 2. β-catenin depletion downregulates fibronectin and LEF1 transcript levels. (A,B) Snai1l increases fibronectin and LEF1 gene expression in LS174T cells. RNAs were extracted from LS174T control cells transfected with Snail1 in a eukaryotic expression vector and analysed by semi-quantitative PCR (A) or qRT-PCR (B) with specific oligonucleotides for the indicated genes. Representative clones are shown in A; the average of the results obtained with three different clones are shown in B. (C) Inducible repression of β-catenin in LS-174T clones. Total-cell protein extracts or RNAs were obtained from clones expressing Snail1 (clones S) or controls (clones C). Doxycycline (1 µg/ml) was added for 6 days prior to the preparation of the extracts as indicated. β-catenin and Snail1 expression were analysed by western blot with specific mAbs. Anti--tubulin was used as a loading control. Endogenous full-length TCF4 mRNA and exogenous TCF4 plus endogenous TCF4 mRNA were also analysed by RT-PCR. As a control, HPRT levels were determined. (D) β-catenin siRNA decreases fibronectin and LEF1 transcript levels. RNA was obtained from the above-mentioned cell clones and levels of fibronectin, LEF1 and Myc were determined by qRT-PCR. As a control, HPRT RNA levels were determined. The figure shows the values of fibronectin, LEF1 and Myc RNA levels determined in the presence of doxycycline and referred to the level of the corresponding RNA in the absence of this drug. The average ± s.d. of two independent experiments performed in duplicate with two representative clones is shown.