FIGURE 3.

Aurora kinases are not required for entry into mitosis and activation of Cdk1. A, HeLa cells were synchronized by double thymidine block/release. Synchronized cells were treated with DMSO as control (A) or a selective Aurora A kinase inhibitor (B) or a selective Cdk1 kinase inhibitor (C) or a combination of Aurora A and Plk1 kinase inhibitors (D) 5 h after release from thymidine block. Mitotic index was analyzed by flow cytometry and mitotic progression was also followed biochemically by Western blotting for phospho-histone H1 and H3, autophosphorylation of Aurora A (Thr(P)-288), Myt1 phosphorylation at Thr-495, Cdc2 tyrosine 15 phosphorylation (Y15) and cyclin B1 and Aurora A protein levels. Total Cdc2 protein was used as loading control. E, shown is mitotic arrest with monopolar spindles of cells treated with a selective Aurora A kinase inhibitor. a, shown is a representative control and a treated HeLa cell showing normal bipolar mitotic spindle in the control and monopolar spindle in the treated cell. Blue = DNA; red = pericentrin staining of centrosome; green = α-tubulin staining. b, shown is the mitotic index of the control and cells treated with a selective Aurora kinase inhibitor, which was used at a concentration shown to selectively inhibit Aurora A kinase activity. Cells were fixed and stained as shown in panel A. At least 500 cells were counted, and mitotic index is expressed as percentage of cells at mitosis. c, shown is a mitotic control and a treated cell showing phospho-histone H3 and Aurora A staining. Blue = DNA; green = α-tubulin; red = phospho-histone H3 (upper) and Aurora A (lower). Note that the Aurora A kinase inhibitor prevented centrosome localization of the Aurora A protein. Bar = 7 μm. F, Cdk1 inhibitor, RO3306 (9 μm), block/release of HeLa cells is shown. Cells were treated with RO3306 for 20 h and then were released from the block by washing 3× with fresh medium without RO3306. Total cell lysates were analyzed by Western blot analysis for mitotic markers and activation of Aurora A kinase. Mitotic extract of cells treated with nocodazole for 20 h was used as a positive control. G, NIH-3T3 cells were similarly treated with RO3306 as described for panel F for Cdk1-dependent activation of Aurora A kinase during G₂/M transition. H, shown is Western blot analysis of a synchronized cell cycle progression in the presence or absence of a selective Plk1 kinase inhibitor, BI2536. HeLa cells were synchronized by double thymidine block, and the Plk1 inhibitor was added 5 h after release from thymidine block. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.