WP1130 inhibits DUB activity in primary B-cell tumors. (A) Top: primary myeloma cells were treated with 5 μM WP1130 for 4 hours before assessing DUB activity as described in the legend for Figure 1. The HA-labeled Usp9x protein is denoted. Middle: total protein levels for Usp9x, Mcl-1, and β-actin were also assessed by immunoblotting. Bottom: after 24 hours, aliquots of the same cells were subjected to staining with annexin V-fluorescein isothiocyanate (FITC)/propidium iodide and analyzed by flow cytometry. Viability of untreated control cells was set at 100%, and relative reduction in survival in WP1130-treated cells is noted below each blot. (B) Plasmacytoma cells from a newly diagnosed patient were treated with 0, 1.25, 2.5, and 5.0 µM WP1130 for 4 hours before measuring DUB activity by HA-UbVS labeling, and the labeled DUBs were visualized by anti-HA immunoblotting. Mcl-1 and actin protein levels were also measured by blotting. Viability of CD138⁺ cells was determined as described in (A). (C) Primary CLL cells were left untreated or were treated with WP1130 as described above. Patient CLL60 cells were treated with the WP1130 concentration indicated before assessing DUB activity and Usp9x, Mcl-1, and β-actin expression by immunoblotting.