, all CPC subunits were dispersed from centromeres in cells blocked in taxol and then exposed to the Haspin inhibitor CHR6494, which is structurally distinct from 5Itu, nevertheless showing similar effects as 5Itu. These results suggest that Haspin is the relevant target of 5Itu with respect to CPC targeting to the centromere. In addition, Haspin inhibitors reduced CPC localization whether cells are blocked in nocodazole or taxol. Loss of Borealin at the centromere was also observed when either 5Itu or CHR6494 was added as cells were entering mitosis in the absence of spindle poisons suggesting that this effect does not rely on spindle disruption. To further investigate the role of pH2AT120 and pH3T3 in CPC localization, FRAP was performed in nocodazole-arrested cells stably expressing full-length Borealin-GFP in the presence or absence of Aurora B, Mps1, or Haspin inhibitors. As expected from previous studies, Borealin-GFP exchanged slowly at centromeres in control cells, recovering to ~40% of pre-bleach levels within a 60 second time-window and a rate constant of 0.02 sec-1 32. Inhibiting Aurora B with ZM447439 or Mps1 with reversine did not significantly change the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850363 kinetics of Borealin-GFP recovery at centromeres. Inhibiting Haspin with 5Itu significantly increased the post-bleach recovery kinetics of Borealin-GFP, which recovered ~85% of pre-bleach levels with a rate-constant of 0.05 sec-1 in a 60 second time-window. These results suggest that stable association of Borealin with inner centromeres is regulated by Haspin Author Manuscript Author Manuscript Author Manuscript Author Manuscript Nat Commun. Author manuscript; available in PMC 2015 October 09. Bekier et al. Page 6 kinase activity, but relatively unaffected by Aurora B or Mps1 inhibitors when cells have entered mitosis. Haspin inhibition reveals a kinetochore-proximal CPC pool Despite a major reduction in all CPC family members in response to 5Itu, residual staining is consistently observed. More detailed confocal imaging revealed that residual Borealin was observed in a kinetochore-proximal location. This pattern was observed in cells blocked either in nocodazole or taxol and whether 5Itu was added before or after cells had entered mitosis. Different kinetochore pairs in the same cell showed variation in the exact pattern of Borealin staining after 5Itu-treatment; however, it was not uncommon to observe very close overlap with kinetochore 
markers. Careful analysis of nocodazole-arrested cells not exposed to 5Itu also revealed some kinetochore pairs with low levels of Borealin at or near kinetochores. In general, this pool was difficult to discern due to high levels of inner centromere staining. Additional studies showed that Aurora B and Survivin could be detected in a kinetochore-proximal location in cells blocked in mitosis with taxol and then exposed to 5Itu. MedChemExpress Nigericin (sodium salt) Furthermore, all four core CPC family members showed a kinetochore-proximal staining pattern after 5Itu treatment that could be modulated by overexpression of truncated forms of Borealin. The kinetochore-proximal pool of Borealin revealed after Haspin inhibition is similar to the localization pattern of residual pH2AT120 18,33. Thus, immunofluorescence was performed in nocodazole-arrested A8GFP#24 cells to assess both pH2AT120 and BorealinGFP localization in the same cells after inhibiting Aurora B, Mps1 and Haspin. A8GFP#24 cells were used due to the availability of monoclonal antibodies to GFP, but not Borealin.