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. 2019 Mar 7;104(10):2061–2074. doi: 10.3324/haematol.2018.211110

Figure 3.

Figure 3.

MEK + histone deacetylase inhibitor induced synergistic apoptosis is mediated by BIM. (A) The RAS mutant human multiple myeloma (MM) cell lines H929 and MM1S were treated with AZD6244/LBH589 for 24 h, then whole-cell lysates were blotted for the indicated proteins. (B) MM1S was electroporated with scrambled siRNA or BIM siRNA and then left untreated or treated with 5 nM LBH589. At 72 h, cell viability was assessed using flow cytometry by analyzing the proportion of annexin−/propidium iodide (PI)− cells, shown as percent of control on the Y-axis. Furthermore, the whole-cell lysates were separated using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to western blotting for the indicated proteins to confirm silencing. Error bars represent the standard error of mean of triplicate experiments. Differences between groups were calculated with the Student t test. **P<0.001. (C) (Upper) H929 and MM1S were treated with AZD6244 (250 nM and 150 nM, respectively) and LBH589 (5 nM) for 24 h. BIM immunoprecipitates were separated using SDS-PAGE and subjected to western blotting to examine BCL-2, MCL-1 and BCL-XL binding patterns. Whole cell lysates (input) were also separated and probed for the indicated proteins. (Lower) Immunoprecipitates from MM1S for BCL-2 and MCL-1 were also separated and probed to examine BIM binding. (D) H929 and MM1S were treated with AZD6244 (250 nM and 150 nM, respectively) and LBH589 (5 nM). BAX and BAK immunoprecipitation was performed and western blotting was used to examine levels of BIM and MCL-1 bound to BAX and BAK. Whole cell lysates (input) were also separated and probed for the indicated proteins. All experiments were performed in triplicate.