Effect of ectopic RHAMM expression. U266 MM cells were transfected with a RHAMM-EGFP construct (▪) or control empty vector (EV, □). (A) Western assay of transfected isogenic cells synchronized by double thymidine block and then immunoblotted for expression of RHAMM, actin, or EGFP. (B) The transfected cells were exposed to increasing concentrations of VX-680 for 72 hours and then assessed for percent inhibition of growth in MTT assays (left panel) and percent apoptosis (right panel, flow cytometric analysis of activated caspase 3 expression). Dark bars are RHAMM transfectants, and open bars are empty vector control cells. Results are means of 3 separate experiments where the SDs were all less than 5% of the mean. Percent of cells demonstrating tetraploidy are shown on top of bars of right panel. The percent inhibition of growth and percent induced apoptosis was significantly greater (P < .05) for RHAMM-transfected cells at all doses of VX-680.