Figure 6.

Gal/VNPT55 disrupts NF-κB and Twist1 transcriptional activity. (a) PC-3 and DU145 cells were treated with gal and VNPT55 for 24 h and 50 or 100 μg of total cell lysates separated on a 10% Tris/glycine gel to analyze p65, p52 and phosphorylated p65 (p-p65). (b) RNA collected from treated PC-3 cells were subjected to quantitative real-time PCR to analyze E-Cadherin, MMP-9, Twist1 and Snail mRNA expression (**p<0.001). (c) Protein expression of Snail, Slug, Twist1, RhoA, vimentin and Vascular endothelial growth factor (VEGF) were evaluated by immunoblot analysis after a 24 h treatment in PC-3 and DU145 cells. (d and e) PC-3 and DU145 cells pre-treated with 10 ng TNF-α for 2 h were subsequently treated with gal/VNPT55 at 10 μM for an additional 24 h. Total cell lysates were subjected to cell fractionation. [PC-3-Nu and DU145-Nu are nuclear fractions; PC-3-Cyt and DU145-Cyt are cytosolic fractions]. LSD1 was used as loading controls for nuclear fractions and lactate dehydrogenase (LDH) for cytosolic fraction controls. Twist1, p65 and p-p65 expression levels were analyzed in the different compartments. (f)PC-3 cells were pre-treated with 10 ng TNF-α for 2 h and then treated with gal/VNPT55 for 24 h. Cells were fixed in 3.7% paraformaldehyde and stained with Twist1 mouse monoclonal antibody and images taken