Effect of RHAMM silencing. AF-10 cells were infected with lentivirus expressing shRNA for RHAMM or a scrambled sequence (Control). After selection of transduced clones in blasticidin, the isogenic cell lines were first immunoblotted for expression of endogenous RHAMM (top). Immunoblot was performed 6 hours after synchronizing cells as described in Figure 5. Isogenic cell lines were then exposed to 0, 100, or 200 nM VX-680 for 72 hours, after which percent inhibition was calculated from growth assays and percent apoptosis was assayed by flow cytometric analysis of activated caspase 3 expression (bottom). Results are means of 3 experiments where SDs were less than 5% of the mean for all groups. The degree of percent inhibition and apoptosis in shRNA-expressing cells (▪) was significantly less (P < .05) than that of control cells (□).