s SB-203580 treated with the Aurora-B inhibitor hesperadin, the level of H3T3ph was reduced to approximately 40% in cells in late G2 phase. When Aurora-A and Plk1 were simultaneously inhibited, the level of H3T3-ph was nearly completely abolished. This synergetic effect indicates that this function of Aurora-A is at least partially independent of Plk1 in promoting H3T3-ph. In prometaphase cells, the reduction of H3T3-ph was weaker than the reduction observed in prophase cells when Aurora-A was inhibited but greater than the reduction observed when Aurora-B was inhibited. These data suggest that Aurora-A modulates H3T3-ph mainly in early mitosis, whereas Aurora-B has a more important role in promoting H3T3-ph in later stages. Consistently, in Aurora-A KO MEF cells, the level of H3T3-ph was clearly reduced in late G2-prophase, whereas this reduction was much weaker in prometaphase cells. Similar result of reduction of H3T3-ph after Aurora-A inhibition was also obtained in human cancer cell line A549. To evaluate the contribution of nuclear-localized Aurora-A to the H3T3-ph in late G2 phase, a nuclear export signal -tagged Aurora-A, kinase deficient Aurora-A and wild type Aurora-A were expressed while endogenous Aurora-A was depleted with shRNA. Centrosome separation was not disturbed when the nuclear accumulation of Aurora-A was blocked by NES-Aurora-A in late G2 phase, suggesting that NES-Aurora-A did not disturb either the Aurora-A kinase function at centrosomes or its general kinase activity. Remarkably, compared with uninduced or WT Aurora-A-transfected cells, in the NES-Aurora-A or KD-Aurora-A positive cells the H3T3-ph signal was dramatically decreased in late G2 phase. Therefore, this result confirmed the hypothesis that kinase activity of nuclear Aurora-A is involved in the efficient H3T3-ph in late G2 and prophase. It is worth mentioning that the ratio of lagging chromosome increases from 1% in WT Aurora-A-expressing cells to over 10% in NESAurora-A-expressing cells and the number of binucleated cells was also increased. These data suggest that nuclear accumulation of Aurora-A in late G2 cells is crucial for the accuracy of chromosome segregation. Aurora-A-mediated H3T3-ph is independent of CPC The level of H3T3-ph was also evaluated using western blotting analysis in synchronized HeLa cells upon Aurora-A inhibition. In G2-phase cells, the level of MPM2 epitope was low and was 
not affected by the Aurora or Plk1 inhibitors, suggesting that the cells were well synchronized and that the mitotic population was not changed upon kinases’ inhibitions. Remarkably, in the lysate of G2 cells, the total H3T3-ph was dramatically reduced by the Aurora-A inhibitor. This inhibitory effect was partially restored in prometaphase cells, which is consistent with the results obtained in immunostaining experiments. Thus these results support the hypothesis that Aurora-A kinase is required for H3T3-ph mainly in late G2 phase and that it contributes much less in prometaphase. Aurora-A triggers Haspin-Aurora-B feedback 8 As both Aurora-A and Aurora-B promote H3T3-ph,, and Aurora-A kinase activity is required for the centromere recruitment of Aurora-B, it is possible that Aurora-A modulates H3T3-ph via recruiting Aurora-B/CPC. If this is the case, constitutively active CPC should rescue the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19822652 H3T3-ph signal upon Aurora-A inhibition. However, in CB-INCENP-expressing cells, the H3T3-ph was as Fazhi Yu et al. 9 low as it was in untransfected cells when Aurora-A was inh