Figure 5. SIRT3 negatively regulates MCL1 stability through deacetylation.

(A) SIRT1, SIRT2, and SIRT3 efficiently deacetylate MCL1 in vitro. Shown is IB analysis of MCL1 acetylation after the in vitro deacetylation reaction and WCLs derived from HeLa cells transfected with the indicated Myc-MCL1 and FLAG-SIRT constructs for MCL1 and SIRT protein purification by FLAG and Myc immunoprecipitates (STAR Methods).

(B) MCL1 interacts with SIRT3 at the endogenous level. Shown is IB analysis of WCLs (input) and anti-MCL1 immunoprecipitates derived from 293T cells.

(C) SIRT3 depletion results in the accumulation of MCL1 protein abundance. Shown is IB analysis of WCLs derived from HeLa cells stably expressing the lentiviral shRNA specific for GFP, SIRT1, SIRT2, or SIRT3.

(D) SIRT3 depletion extends MCL1 protein half-life. HeLa cells stably expressing the lentiviral shRNA specific for GFP or SIRT3 presented in (C) were treated with 100 μg/mL CHX for the indicated period before harvesting.

(E) SIRT3 depletion results in impairment of MCL1 poly-ubiquitination. HeLa cells stably expressing the lentiviral shRNA specific for GFP, SIRT1, SIRT2, or SIRT3 were transfected with Myc-MCL1 and His-Ub constructs. 36 h after transfection, cells were treated with MG132 (10 μM) overnight before harvesting.

(F) SIRT3 depletion accumulates endogenous Ac-K40-MCL1. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from HeLa cells stably expressing the indicated lentiviral shRNA specific for GFP or SIRT3.

(G) Ectopic SIRT3 expression increases ubiquitination of WT MCL1 but not K40R. 293T cells were transfected with Myc-MCL1, FLAG-SIRT3, and His-Ub constructs as indicated. 36 h after transfection, the cells were treated with MG132 (20 μM) for 5 h before harvesting. Left: His-Ub-conjugated proteins were captured with Ni-NTA agarose beads. Right: a schematic model of SIRT3-mediated MCL1 ubiquitination through K40 deacetylation followed by USP9X dissociation.

(H and I) SIRT3 depletion confers resistance to doxorubicin-induced apoptosis through MCL1 stabilization. CRISPR-Cas9-mediated MCL1 KO and its parental HeLa cells were infected with the lentiviral shRNA specific for GFP or SIRT3. These cells were treated with the indicated concentrations of doxorubicin for 24 h and then subjected to IB analysis (H) and a cell viability assay (I). Data are presented as mean ± SD; n = 3 biological replicates; **p < 0.01, ***p < 0.001.

Data in (A)–(H) are representative of at least two independent experiments. See also Figure S5.