NPI-0052 inhibited migration and adhesion of BCWM.1 cells in vitro and homing in vivo. (A) Transwell migration assay showing inhibition of migration of BCWM.1 cells and primary CD19⁺ cells in the presence of NPI-0052 (2.5-20 nM), bortezomib (10 nM), or NPI-0052 (10 nM) in combination with bortezomib (10 nM). SDF-1 30 nM was placed in the lower chambers and induced migration compared with control with no SDF-1 (Ctrl, control). SDF-1 was placed in the lower chambers of the NPI-0052/bortezomib–treated wells. (B) Adhesion assay with BCWM.1 cells and primary CD19⁺ cells in the presence or absence of NPI-0052 (10 nM), either alone or in combination with bortezomib (10 nM). BCWM.1 cells demonstrated increased adhesion in fibronectin-coated wells (control) compared with BSA-coated wells (BSA, bovine serum albumin). All data represent mean (± SD) of triplicate experiments. (C) BCWM.1 cells were cultured with control media or NPI-0052 (10 nM) with and without bortezomib (10 nM) for 4 hours, in the presence or absence of fibronectin (FN). Nuclear extracts were subjected to Western blotting using anti–p-p65, -p50, and -nucleolin antibodies. (D) BCWM.1 cells were cultured with control media or NPI-0052 (10 nM), with and without bortezomib (10 nM) for 4 hours, in the presence of fibronectin. Whole-cell lysates were subjected to Western blotting using anti–p-FAK, anti-ILK, and anti–α-tubulin. (E) In vivo flow cytometry. DiI-labeled cells treated with bortezomib (B) and NPI-0052 (N) and DiR-labeled untreated cells were injected in the tail vein of 2 BALB/c mice. Cells were counted every 5 minutes for 45 minutes, as described in “Methods.”