Inhibition of Aurora-A kinase (Thr288) phosphorylation in MM cells by MLN8237. (A) Expression of Aurora-A kinase protein in a panel of MM cell lines by immunoblotting of equal amounts of total protein (30 μg) with anti–Aurora-A kinase antibody. (B) Immunoblotting with anti–phospho (Thr288)-Aurora-A kinase antibody showed inhibition of Aurora-A kinase autophosphorylation (Thr288) in MM1.S and OPM1, with or without synchronizing with nocodazole. Expression of phospho (Thr288)-Aurora-A kinase protein was shown relative to total expression of Aurora-A kinase protein. Actin was used as a loading control. MM1.S (C) and OPM1 (D) MM cell lines were treated with DMSO or MLN8237 (0.5μM) for 24 hours and then stained with anti–phospho (Thr288)-Aurora-A kinase antibody (red), αtubulin (green), and DNA (blue). Overlapping localization is shown in the merged images. Arrow indicates Aurora-A autophosphorylation on Thr288 in the centrosome (original magnification ×40). Magnified single mitotic cell image is shown in the right panel. The number of mitotic cells with phosphorylated Aurora-A kinase (pT288) relative to total number of mitotic cells are shown. (E) To assess the effect of MLN8237 on Aurora-B kinase and mitosis, MM1.S and OPM1 cells were treated for 1, 24, and 48 hours with DMSO or MLN8237 (0.5μM). Flow cytometric (top panel) and Western immunoblot (bottom panel) analyses in representative MM cell lines show mitotic cells and active Aurora-B with increased DNA, costained with phospho-(Ser10)-histone H3 (Alexa Fluor 488) and PI.