E-cadherin controls β-catenin and NF-B transcriptional activity in mesenchymal gene expression

JCS021667 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Table S1. Oligonucleotides used for quantitative and semiquantitative RT-PCR and PCR.

• Supplemental Figure S1 -

  Fig. S1. Concomitant induction of fibronectin and repression of E-cadherin by ectopic Snail1 expression. RNAs were extracted from HT-29 M6 cells transfected with Snail1 under the control of tet-off system and analysed by RT-PCR and Southern blot. Incubation of these cells without doxycycline (dox) induces the expression of the Snail1 transgene. At day 0, 2 µg/ml of doxycycline was added to the medium to turn-off Snail expression. The figure shows a representative experiment of three performed.

• Supplemental Figure S2 -

  Fig. S2. Activity of the LEF1 promoter, but not fibronectin promoter, is sensitive to TCF-4/β-catenin activity. RWP1 cells were co-transfected with TOP plasmid (25 ng), pGL3-LEF1 promoter (100 ng) or pGL3-fibronectin promoter (100 ng) and pcDNA3 containing VP16-TCF-4 (5 ng), ΔTCF-4 (250 ng), or empty vector as a control, and 1 ng of pRL-SV40. Luciferase activity was assayed after 48 hours of expression. Graphics show the promoter activity induced by the indicated Wnt pathway effectors relative to the basal promoter activity in presence of empty pcDNA3. The error bars indicate standard deviation.

• Supplemental Figure S3 -

  Fig. S3. β-catenin-depletion affects β-catenin dependent signalling in Ls174T cells. (A) Ls174T siRNA β-catenin cells grown on glass coverslips were treated with or without doxycycline (1 µg/ml) for 6 days. Cells were fixed with 4% PFA and analysed by immunofluorescence with a β-catenin mAb. (B) Ls174T control, β-catenin siRNA and ΔTCF4 mRNA teton clones were co-transfected with 250 ng of TOP plasmid and 1 ng of pRL-SV40. Luciferase activity was assayed after 48 hours of expression in the presence or absence of 2 µg/ml of doxycycline. Graphics show the promoter activity relative to the basal promoter activity in the absence of doxycycline. The error bars indicate standard deviation. Equivalent results were obtained in the same clones cotransfected with 25 ng of a pcDNA-Snail-HA vector.

• Supplemental Figure S4 -

  Fig. S4. Fibronectin and LEF1 promoters are sensitive to VP16-Rel. The activity of fibronectin and LEF1 promoters was assayed in SW-480 cells co-transfected with increasing amounts (10 and 25 ng) of VP16-Rel expression plasmid. The figure shows the average ± s.d. of three experiments performed in triplicate.

• Supplemental Figure S5 -

  Fig. S5. Localisation of p65 and E-cadherin in IEC-18 cells. The cellular distribution of p65 and E-cadherin was determined by immunofluorescence as previously described. When indicated cells were treated with TNF-α (20 ng/ml) for 6 hours prior to the analysis, and washed with PBS plus 0.025% NP-40 prior to fixation to decrease cytosolic staining. A detail of the area indicated is shown at right panels. The arrows point to areas where presence of p65 in the membrane was more clearly shown.