Figure 2.

Inhibition of E-cadherin expression in sparse cultures by Slug. (A) SW480 cells were seeded at: lane 1, 6 × 10³ cells/cm²; lane 2, 2 × 10⁴ cells/cm²; lane 3, 4 × 10⁴ cells/cm²; and lane 4, 8 × 10⁴ cells/cm²), and the level of E-cadherin, Slug, Snail, and GAPDH poly(A)-RNA was determined by Northern blot hybridization with ³²P-labeled cDNA probes. (B) Cells from a confluent dish were seeded as sparse or dense cultures (as in Fig. 1 A), and at different times the level of Slug was determined by Western blot analysis of adherent cells using equal amounts of total protein. (C) Dense cultures were transfected with an E-cadherin promoter reporter and 14 h later the cells were seeded as sparse and dense cultures and promoter activity determined at different times after plating. (D) Sparse and dense cultures were transfected with WT mouse E-cadherin promoter reporter (wt), an E-box mutant promoter (mE-box), with or without a Slug cDNA plasmid and luciferase activity was determined. (E) Human and mouse WT E-cadherin promoter reporters were transfected into 293-T cells in the presence and absence of Slug and luciferase activity determined. (C–E) The error bars represent the mean ± SD from triplicate plates. (F) SW480 cells were transfected with a plasmid coding for both GFP and Slug, or histone-GFP, and stained for E-cadherin with rhodamine-labeled secondary antibody. Note that Slug expression correlated with reduced E-cadherin level, it inhibited the WT E-cadherin promoter and reduced E-cadherin expression in transfected cells. The arrows point to E-cadherin in adherens junctions of histone-GFP transfected cells. Bar, 10 μm.