Figure 2. p300-mediated MCL1 acetylation leads to MCL1 stabilization through decreasing ubiquitination.

(A) p300 knockdown decreases MCL1 protein abundance. Shown is IB analysis of WCLs derived from HeLa cells infected with the indicated lentiviral shRNA specific for GFP or p300.

(B) p300 knockdown shortens MCL1 protein half-life. Shown is IB analysis of WCLs derived from HeLa cells stably expressing the indicated lentiviral shRNA. The cells were treated with the protein synthesis inhibitor cycloheximide (CHX; 100 μg/mL) for the indicated periods before harvesting.

(C) Quantification of the MCL1 band intensities of IB replicates in (B). Data are presented as mean ± SD; n = 3 independent experiments, *p < 0.05.

(D) Treatment with the p300/CBP inhibitor A-485 shortens MCL1 protein half-life. Shown is IB analysis of WCLs derived from HeLa cells. Cells were pretreated with A-485 (3 μM) overnight and then treated with 100 μg/mL CHX for the indicated periods before harvesting.

(E) Quantification of the MCL1 band intensities of IB replicates in (D). Data are presented as mean ± SD; n = 3 independent experiments, **p < 0.01.

(F) HeLa cells stably expressing the indicated lentiviral shRNA specific for GFP or p300 were transfected with His-tagged ubiquitin (His-Ub) and Myc-MCL1. 36 h after transfection, the cells were treated overnight with MG132 (10 μM) before harvesting. His-Ub-conjugated proteins were captured with Ni(2+)-nitrilotriacetic acid (Ni-NTA) agarose beads and subjected to IB analysis.

(G) Acetylation-mimetic K40Q substitution results in decreased poly-ubiquitination of MCL1 in cells. 293T cells 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.

(H) Ectopic p300 expression decreases poly-ubiquitination of WT MCL1 but not K40R. 293T cells were transfected with the indicated constructs. 36 h after transfection, cells were treated with MG132 (20 μM) for 5 h and harvested for the Ni-NTA pull-down.

(I) Treatment with the p300 inhibitor A-485 induces poly-ubiquitination of the WT but not the K40R mutant form of MCL1. 293T cells were transfected with the indicated constructs. 24 h after transfection, cells were treated with or without A-485 (3 μM) for 24 h and MG132 (10 μM) for 10 h before harvesting.

(J) Acetylation-mimetic K40Q extends MCL1 protein half-life. Shown is IB analysis of WCLs derived from HeLa cells transfected with the indicated Myc-MCL1 constructs. 48 h after transfection, cells were treated with 100 μg/mL CHX for the indicated periods before harvesting.

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

(L) Ectopic p300 expression extends the protein half-life of the WT but not the K40R form of MCL1. Shown is IB analysis of WCLs derived from 293T cells transfected with Myc-MCL1 and HA-p300 constructs as indicated. 48 h after transfection, cells were treated with 100 μg/mL CHX for the indicated periods before harvesting.

(M) Quantification of the Myc band intensities of IB replicates in (L). Data are presented as mean ± SD; n = 3 independent experiments, **p < 0.01; NS, not significant.

Data in (A) and (F)–(I) are representative of at least two independent experiments. See also Figure S2.