Figure 8 (IMAGE)
Caption
BI-D1870 in combination with vincristine increase metaphase arrest and apoptosis synergistically. (A, B) Cell cycle distribution of HL60 and Nalm6 cells treated with BI-D1870 and vincristine. Cells were treated with BI-D1870 (2.5 μM) and/or vincristine (5 nM) for 1 d, and then cells were fixed and analyzed for cell cycle distribution. Cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and antibodies. Percentages of live cell populations at each cell cycle phase determined by DNA content (DAPI) and levels of p-H3, Cyclin A, and Cyclin B were graphed. (C) Cells were treated with BI-D1870 (2.5 μM) and/or vincristine (5 nM) for 1d or 2d. Percentages of Annexin V-positive apoptotic cells are calculated. (D) Synergistic combination effect of BI-D1870 and vincristine. Cells were treated with various concentrations of BI-D1870 and vincristine for 3d. Viability was assessed using the CellTiter glo assay kit. The combination index (CI) values were calculated by the Chou-Talalay method using CalcuSyn software. CI > 1: antagonism, CI = 1: additive Effect, CI < 1: synergism. Data are the mean ± SEM (n = 4). (E) BI-D1870 in combination with vincristine-induced apoptosis synergistically in primary patient AML cells. Primary cells were treated with BI-D1870 (1.5 μM) and/or vincristine (3 nM) for 2d, and then Annexin V-positive cells were assessed using flow cytometry. Data are graphed as mean ± SEM (n = 3). **p < 0.01; ***p < 0.001. (F) A proposed action model of the RSK inhibitor BI-D1870 in mitotic exit. BI-D1870 inhibits the disassembly of the MCC to activate APC/C for anaphase transition by blocking the phosphorylation-induced conformation change of MAD2.
Credit
Correspondence to - Kathleen M. Sakamoto - kmsakamo@stanford.edu
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