Usp9x KD reduces myeloma cell survival and proliferation and upregulates Usp24. (A) Immunoblot confirming Usp9x and Mcl-1 KD in H929 and MM1.S myeloma cells. Usp9x or Mcl-1 were knocked down by control or specific shRNA retrovirus and were selected for puromycin resistance before cell lysates were examined for the protein indicated (left) or cell survival by measurement of annexin positivity (right). The results represent the average ± standard deviation (SD) of triplicate assays. (B) Top: equal numbers of scrambled control and Usp9x KD cells were plated on day 0, and cell numbers were assessed daily for 3 days. The numbers represent the average ± SD of triplicate cell counts. Bottom: cell cycle analysis was performed on control (red bars) and Usp9x KD (blue bars) cells obtained at day 3 following initial plating. Cell cycle phase was determined by propidium iodide staining and flow analysis, and each value represents the average ± SD of triplicate assessments. P values are shown above each pair. (C) DUB activity was assessed (as described in the legend for Figure 1) in control KD and Usp9x KD H929 cells left untreated or treated with 5 μM WP1130 for 4 hours. Usp9x, Mcl-1, PARP, and actin were also assessed by immunoblotting. HA-UbVS–labeled Usp9x and Usp24 are denoted (migration at similar molecular size). (D) Lysates from control (Con) and chronic Usp9x KD myeloma cells were subjected to immunoblotting for Usp9x, Usp24, and actin. (E) Colon cancer cell lines with (Usp9x^−/−) or without (Usp9x^+/+) full Usp9x gene disruption were assessed for DUB activity by HA-UbVS labeling (top) of equal protein lysates. Lysates were also probed for Usp9x, Usp24, and β-actin protein expression. (F) Equal protein lysates from myeloma cells were subjected to immunodepletion of the DUB indicated with anti-Usp9x or Usp24 (or control antibody [Ab]) before measurement of total DUB activity by HA-UbVS labeling. The resultant loss of Usp9x or Usp24 from each supernatant was determined by immunoblotting. (G) Usp9x (left) and Usp24 (right) gene expression in myeloma patient samples was assessed by quantitative polymerase chain reaction and compared with H929 control KD and Usp9x KD cells (green bars). Samples designated as high risk (red bars) or good risk (blue bars) of progressive disease based on their cytogenetic profiles are denoted. Each bar represents the average ± SD of triplicate samples. (H) Usp9x and Usp24 protein expression was assessed by immunoblotting cell lysates derived from myeloma (H929) and MCL (Z138) cell lines, normal tonsilar B cells, or samples derived from two newly diagnosed or two relapsed myeloma patients. Actin was blotted as a protein loading control. (I) Tissue microarrays from normal tissue (spleen and tonsil) and confirmed pathologic samples from MCL (left) or CLL (right) were subjected to immunostaining for Usp9x and Usp24. Examples of tumor samples with intermediate and high expression in the same specimen are shown. The Usp9x/Usp24 staining pattern in additional samples is shown in supplemental Figure 2. **P < .01; ***P < .005.