In vitro functional effect of ST3GAL6 knockdown in MM cell lines. (A) Effect of ST3GAL6 knockdown on the ability of MM1S and RPMI-8226 cells to adhere to BMSCs isolated from the BM of MM patients. MM1S clone A2, P = .0263; clone A3 P = .0286. RPMI-8226 clone A2, P = .0023, clone A3 P = .003. (B) Adhesion of ST3GAL6 knockdown cells to fibronectin in vitro compared with corresponding scrambled control cells. MM1S A2 clone, P = .0003; RPMI-8226, P < .0001. Similar results were obtained for MM1S and RPMI-8226 A3 clones (data not shown). (C) Adhesion of MM1S and RPMI-8226 shST3GAL6 cells to HUVECs compared with scrambled control cells. Both shST3GAL6 knockdown cells had a significantly reduced ability to adhere to HUVECs in vitro. MM1S clone A2, P = .0003; clone A3 P < .0001. RPMI-8226 clone A2, P = .01; clone A3 P = .003. (D) Migration of ST3GAL6 knockdown cells to BMSC-conditioned media (BMSC CM) in a transendothelial migration assay. The ability of ST3GAL6 knockdown cells to achieve transendothelial migration was significantly reduced compared with scrambled control cells. MM1S clone A2, P < .01; clone A3, P < .01. RPMI-8226 clone A2, P = .0003; A3 P = .0001. (E) Western blot analysis demonstrating reduced levels of adhesion-related proteins in MM1S shST3GAL6 cells that were co-cultured with the BMSCs from MM patients for 24 hours. Phosphorylated paxilin was reduced in shST3GAL6 compared with scrambled control cells cultured in the presence of BMSCs. Phosphorylation of cofilin was greater in the MM cells co-cultured with BMSCs. We also noted an induction of P-Src in scrambled control cells upon co-culture with BMSCs, which was not apparent in the shST3GAL6 cells. α-tubulin was used as a loading control. (F) Rolling of shST3GAL6 cells on P-selectin compared with scrambled control cells demonstrates a reduced ability of these cells to roll on P-selectin (P = .02).