Figure 5. Knockdown or overexpression of SLUG modulates E-cadherin and P-cadherin adhesion molecules.

(A) Expression levels of EMT markers in SLUG-depleted cells: 501mel cells were transfected with control siRNA (siCTRL) for 4 days or two SLUG siRNAs (siSLUG #1 and #2) for the indicated times. Expression levels of SLUG, E-cadherin, P-cadherin and Fibronectin were analyzed by immunoblotting. HSP60 was used as loading control. (B) Morphology of SLUG-depleted cells: expression of SLUG (green), E-cadherin (cyan) and actin cytoskeleton (Texas Red-X phalloidin) following siRNA-mediated SLUG depletion in 501mel cells was analyzed by fluorescence staining and confocal microscopy. Bars, 10 µm. (C) Ectopic SLUG expression induces an EMT-like phenotype: control (bl. CTRL) or SLUG-overexpressing (bl. SLUG #1 and #2) 501mel cell populations were analyzed by immunoblotting for expression of SLUG, E-cadherin, P-cadherin, Fibronectin and HSP60 (loading control). (D) Depletion of SLUG increases Ca²⁺-dependent cell-cell adhesion: adhesion assays were performed as described in the Materials and Methods after treatment of 501mel cells with siCTRL or siSLUG as indicated. The phase-contrast pictures show aggregates formed in presence of 1 mmol/L CaCl₂ alone or with 3 mmol/L EDTA. The average of two independent adhesion assays and SD are presented. Columns, average of two independent assays; error bars, SD. *P<0.05 (Student’s test). (E) SLUG regulates E-cadherin mRNA levels: RNAs were prepared from 501mel cells transfected with siCTRL or siSLUG for 4 days, and from bulk selected control or SLUG-overexpressing 501mel cells. The relative mRNA expression levels of SLUG and E-cadherin were measured by SYBR green-based real-time Q-PCR. Columns, mean of three independent amplifications performed in duplicate; error bars, SD. *P<0.05 (Student’s test). (F) E-cadherin promoter activity: 501mel cells were transfected with siCTRL, siSPARC or siSLUG, and 24 hours later with wild-type E-cadherin promoter reporter construct (left). 501mel cells were co-transfected with an empty vector (mock) or vectors expressing SPARC or SLUG and wild-type (−178 wt/luc) or mutant (mE-pal/luc) E-cadherin promoter reporter constructs (right). After 3 days, luciferase activities were measured and normalized to β-galactosidase activities. Columns, mean of triplicates; errors bars, SD.