Figure 2
Geminin interacts with TopoIIα on chromosomes in HME cells. (A) HME cells synchronized in G0/G1, S, G2/M and M/G1 were sonicated and immunoprecipitated with the indicated antibodies, followed by immunoblotting with the indicated antibodies. (B) HME cells treated with etoposide (10 μM, overnight) were then collected and processed for analysis using trapped in agarose DNA immunostaining (TARDIS) assay. Shown are examples of cells costained with Hoechst 33258 blue, TopoIIα and geminin antibodies (1 to 3), costained with Hoechst 33258 blue, TopoIIα but not Cdc7 antibodies (4 to 6) or costained with Hoechst 33258 blue, geminin but not Cdc7 antibodies (7 to 9). Geminin-silenced HME cells (10), plus cell division cycle 7 silencing (siCdc7) (11) or plus casein kinase Iε (CKIε) overexpression (12) are shown. TopoIIα-silenced HME cells (13) plus Cdc7-silenced (14) or plus CKIε overexpression (15) are also shown. (C) Quantification of cells costained with Hoechst 33258 blue and Cdc7 (white bars), geminin (red bars) or TopoIIα (black bars) in TARDIS assays 72 hours after control, Cdc7, geminin, TopoIIα, geminin silencing and Cdc7 silencing (or CKIε overexpression); geminin and TopoIIα silencing; or TopoIIα silencing and Cdc7 silencing (or CKIε overexpression). Values presented are means ± SD. **P ≤ 0.01. (D) In vitro kinase assay performed on purified TopoIIα using CKIε or Cdc7 immunoprecipitated from HME cells.