Figure 4. MCL1 acetylation promotes interaction with USP9X, resulting in MCL1 deubiquitination and stabilization.

(A) Acetylation-mimetic MCL1 K40Q enhances the interaction between MCL1 and USP9X. Shown is IB analysis of WCLs and anti-FLAG immunoprecipitates derived from 293T cells transfected with the indicated constructs.

(B) USP9X depletion reverses the ubiquitination levels of acetylation-mimetic MCL1 K40Q. HeLa cells stably expressing the indicated lentiviral shRNA were transfected with the indicated Myc-MCL1 and His-Ub constructs. 36 h after transfection, cells were treated overnight with MG132 (10 μM) before harvesting.

(C) Treatment with the USP9X inhibitor reverses ubiquitination of acetylation-mimetic MCL1 K40Q. HeLa cells were transfected with the indicated Myc-MCL1 and His-Ub constructs. 36 h after transfection, the cells were treated with MG132 (10 μM) in the presence or absence of WP1130 (5 μM) for 4 h before harvesting. Left: His-Ub-conjugated proteins were captured with Ni-NTA agarose beads. Right: quantification of the Myc-poly-ubiquitination band intensities of IB replicates. Data are presented as mean ± SD, n = 3 independent experiments, *p < 0.05, **p < 0.01.

(D) USP9X depletion abolishes stabilization of MCL1 K40Q. HeLa cells stably expressing the lentiviral shRNA specific for GFP or USP9X were transfected with the indicated Myc-MCL1 constructs. 36 h after transfection, cells were treated with 100 μg/mL CHX for the indicated periods before harvesting.

(E) Quantification of the Myc band intensities of IB replicates in (D). Data are presented as mean ± SD; n = 3 independent experiments, **p < 0.01.

(F and G) Treatment with the USP9X inhibitor WP1130 efficiently downregulates acetylation-mimetic MCL1 K40Q. Shown is IB analysis of WCLs derived from the MCL1-reintroduced CRISPR-Cas9-mediated MCL1 knockout (KO) HeLa (F) and HCT116 (G) cells presented in Figure 3. These cells were treated with WP1130 (10 μM) for the indicated periods before harvesting.

(H and I) Treatment with the USP9X inhibitor WP1130 abrogates the anti-apoptotic effect of acetylation-mimetic MCL1 K40Q. These cells were treated with the indicated concentrations of WP1130 for 24 h and then subjected to cell viability assays. Data are presented as mean ± SD; n = 3 biological replicates.

Data in (A), (B), (F), and (G) are representative of at least two independent experiments. See also Figure S4.