WP1130 affects cellular DUB activity. (A) Lysates (5 μg) from untreated (control) or WP1130-treated (5μM, 4 hours) cells were incubated with 1 μg of K48-linked (left) or K63-linked (right) free chains of polyubiquitin (Ub1-5) for 10 minutes at 37°C. The extent of free chain hydrolysis by active DUB in the each lysate was examined by ubiquitin immunoblotting. Ubiquitin polymer standards (Ub1-5) were resolved on the left and actin was immunoblotted as a protein-loading control. (B) K562 cells treated with 5μM WP1130 (0 to 8 hours) were lysed in DUB-labeling buffer as described in “Ub-AMC protease assay.” Clarified supernatant (20 μg) was incubated with 200nM HA-UbVs (Boston Biochem) for 1 hour at 37°C. HA immunoblotting was used to assess changes in DUB activity (top). Tubulin was probed as a protein-loading control (bottom). The level of the upper HA-labeled band (arrow) was reduced by WP1130 and was determined to represent Usp9x (molecular weight, 293 kDa). The blot was reprobed for Usp9x as a measure of its protein-loading level (middle blot). (C) Usp9x was immunoprecipitated from K562 cells, washed, and incubated in DUB assay buffer containing NEM (5mM), WP1130 (5μM), or DMSO in a 100-μL reaction volume for 30 minutes at 37°C in 96-well fluorometry plates. K562 cell lysates immunoprecipitated with rabbit IgG were used as a control. After incubation, 500nM Ub-AMC was added to the reaction and the release of AMC-fluorescence was recorded over time. The representative fluorescence change over time is shown. The Usp9x activity results represent the average ± SD of triplicate assays. Similar results were obtained in 2 additional independent experiments. (D) K562 and K562R cells were treated with WP1130 for the intervals noted before total cell lysates were subjected to Mcl-1 and actin immunoblotting. (E) In vivo K562 cells were left untreated or treated with 5μM imatinib, 0.5μM dasatinib, 0.5μM TG101209, or 5μM WP1130 for 4 hours before cell lysates were analyzed for Usp9x activity by incubation with HA-UbVs and HA immunoblotting (as described in panel B). In vitro K562 cell lysates were treated with DMSO (control) or 5μM WP1130 for 30 minutes at 37°C before assessing Usp9x activity by HA-UbVs labeling followed by HA blotting. The top portion of the HA immunoblot containing Usp9x is shown. The membrane was stripped and immunoblotted for Usp9x and actin. (F) WDT-2 cells were treated with WP1130 alone or pretreated with imatinib (5μM), dasatinib (0.5μM), or TG101209 (0.5μM) for 1 hour before additional incubation in the presence of WP1130. All cells were harvested 4 hours after WP1130 treatment and total cell lysates were subjected to Mcl-1 immunoblotting. Actin was immunoblotted as a protein-loading control (Note: more protein was loaded in samples obtained from TG101209-treated cells). (G) Left inset, BV-173 cells were untreated (Control) or infected with LVX-shRNA (shRNA-Con) or LVX-shRNA-Usp9x (shRNA-Usp9x) as described in “shRNA and Usp9x silencing.” After puromycin selection, Usp9x and Mcl-1 levels were examined by immunoblotting. Actin was immunoblotted as a protein-loading control. Left, Control and shRNA expressing BV-173 cells (as indicated) were treated with the indicated concentration of imatinib for 72 hours before assessing viability by MTT staining. The results represent the average ± SD of 4 replicates. Statistical significance was determined with a paired Student t test. *P < .05 for shRNA-Usp9x compared with Control or shRNA-Con. Right, BV-173 cells (control and shRNA-expressing as noted) were treated with the indicated concentration of ABT-263 for 72 hours before assessing viability as described in the center panel. The results represent the average ± SD of 4 replicates. Statistical significance was determined with a paired Student t test. *P < .05 for shRNA-Usp9x compared with Control or shRNA-Con.
