Immunoprecipitated proteins Tofacitinib CP-690550 were then incubated for 30 minutes with 100 or 10 ng of purified TopoIIa in the presence of radioactive ATP. While mock and Sp1 immunoprecipitates did not phosphorylate TopoIIa in this assay, Cdc7 phosphorylated the 100 ng quantity, and CKI�� phosphorylated the 100 and 10 ng quantities, of purified TopoIIa protein. These data suggest that Cdc7 indeed phosphorylates TopoIIa, at least in vitro. Cdc7 is upregulated in geminin silenced cells to enforce the mitotic checkpoint induced in these cells Neither Cdc7 silencing nor CKI�� overexpression affected chromosome segregation in control HME GFP H2B cells or HME cells. Geminin but not TopoIIa silencing increased Cdc7 and decreased CKI�� expression.

Cdc7 silencing or CKI�� overexpression in geminin silenced cells restored chromosome segregation Inhibitors,Modulators,Libraries stalled in gemi nin and not in TopoIIa silenced HME cells expressing H2B GFP or in HME cells. These data reinforce the view that CKI�� is a positive regulator and Cdc7 is a negative regulator of TopoIIa chromosome localization and segregation function. Geminin, TopoIIa and Cdc7 silencing or CKI�� overexpression had minimal effects on S phase progression Inhibitors,Modulators,Libraries or DNA replication based on cell cycle and bromodeoxyuridine incorporation analysis. Moreover, while Cdc7 silencing or CKI�� overex pression did not block cells from existing mitosis, gemi nin or TopoIIa silencing did, which can explain why these treat ments had no effect on HME cell viability or cell death. However, Cdc7 cosilencing or CKI�� overex pression in geminin and not TopoIIa silenced cells reduced cell viability and induced cell death.

These data suggest that restoring TopoIIa localization and function by silencing of Cdc7 or overexpression of CKI�� in geminin silenced and or mitosis arrested cells induces cell cycle progression followed by cell death. Presumably, cells are unprepared Inhibitors,Modulators,Libraries to complete mitosis, perhaps because of the low expression of mitotic pro teins observed in these cells. Finally, we speculate that the cell death observed in geminin and TopoIIa silenced cells is due to the activation of p53 in these G2 M arrested cells. These data put Cdc7, like CKI��, upstream of TopoIIa and both Cdc7 and CKI�� downstream of geminin with regard to chromosome segregation and mitosis progression.

Cdc7 upregulation in geminin silenced cells suppresses TopoIIa chromosome localization and decatenation activity Next Inhibitors,Modulators,Libraries we studied whether geminin is required for TopoIIa decatenation activity. TopoIIa was immuno precipitated Inhibitors,Modulators,Libraries from the chromatin of HME cells silenced from Cdc7, geminin or TopoIIa for 72 hours or exposed to 10 uM doxorubicin or 10 uM etoposide for 24 hours. To confirm that chromatin bound TopoIIa was used in these experiments, immu noprecipitated proteins were digested with proteinase K and the DNA was visualized Y-27632 FDA on agarose gel.