Figure 3. siRNA-mediated knockdown of subunit a3 in breast cancer cells reduces in vitro migration.

A. MB231 cells were incubated with an siRNA pool specific for subunit a3 or scrambled siRNA (‘control’). After 72 hours, cells were lysed and protein lysates separated by SDS-PAGE on 4-15% gradient acrylamide gels followed by transfer to nitrocellulose. Immunoblotting was performed using monoclonal antibodies against subunit a3 and tubulin. The blot displayed is representative from three separate siRNA experiments. Western blots from each experiment were quantitated and the ratio of subunit a3 expression in control versus a3 siRNA-transfected cells was calculated. The graphed data represent the average degree of a3 knockdown relative to control cells. *, p < 0.05 relative to control cells. B. Control and a3 siRNA-transfected cells were treated in the presence or absence of 100 μM of the V-ATPase specific inhibitor concanamycin A, plated onto FluorBlok™ inserts and allowed to migrate towards a chemoattractant on the trans-side of the well for an average of 20 hours. Cells were then stained with Calcein AM and the number of cells that had migrated to the trans-side were counted, with 3 wells analyzed per sample and an average of 10 images analyzed per well. Values are the mean of three independent siRNA experiments and expressed relative to control untreated cells. All error bars indicate S.E. *, p < 0.05 relative to control untreated cells.