Figure 5
Biochemical analysis of cdc42 activated form in A2780 ovarian cancer cells over-expressing eIF6. A) Measurement of cdc42 activity analyzed by GST-PAK1 p21-binding domain pull-down. The figure shows one of three independent experiments with similar results. B) We treated A2780 cells (2.5 × 105 per well) with the molecular probe ML 141 at the indicated concentrations for 72 hours. Mock treatments were carried out treating the cells in the same medium with DMSO 0,1%. Cells migrated in the lower chamber were stained with crystal violet dye. In the lower chamber, medium supplemented with 10% FBS was used as chemoattractant and also in this chamber the molecular probe was added at the concentration used in the upper chamber. The histograms are plotted as mean ± S.D. They represent the averages of three independent experiments with a P-value < 0.05 (**) calculated with the t-test. C) ML 141 did not show cytotoxicity in A2780 cell lines. The sensitivity was determined counting the number of cell viability by Trypan Blue exclusion staining. A2780 cells were treated with ML 141 10 μM or DMSO 0,1%. Cell viability was determined by trypan blue dye exclusion assay at the indicated time after ML 141 addition. The histograms represent the average of unstained cells and they are presented as mean ± S.D. The results assess three independent experiments. D) Enhanced migration of A2780 cells induced by eIF6 over-expression with respect the control cells was decreased in presence of cdc42 inhibitor ML 141. In particular, both control (pcDNA3.1) and eIF6-overexpressing (pcDNA3.1-eIF6) cells were affected in their migratory capacity by ML 141. However, the effect was more pronounced on A2780 cells over-expressing eIF6 for the synergistic effect of the inhibitor on both the intrinsic migratory capacity of the cells (as shown by the control) and the eIF6-induced motility.