Figure 5

Geminin overexpression inhibits TopoIIα chromatin localization and activity. (A) Expression of Cdc7, CKIε, geminin and TopoIIα in several breast cancer cell lines. (B) Top: TopoIIα immunoprecipitated from the chromatin of MDAMB231 cells following Luc (control) or geminin silencing for 72 hours and treatment with dimethyl sulfoxide (DMSO), etoposide (10 μM), PHA767491 (10 μM) or IC261 (10 μM) during the last 24 hours. Bottom: TopoIIα and geminin from whole cell extracts of the treatments described above. (C) Quantification of decatenation using k-DNA as the substrate by TopoIIα immunoprecipitated from the chromatin of MDAMB231 treated with the treatments indicated in (B). Values presented are means ± SD. **P ≤ 0.01. (D) TopoIIα levels on the chromatin of HME or induced Gem9 (72 hours) following 24-hour exposure to etoposide (etopo, 10 μM) and doxorubicin (doxo, 10 μM). (E) The effect of DMSO (none) or 10 μM etoposide, doxorubicin, ICRF-187 or ICRF-193 exposure for 24 hours on the viability of Gem9 cells grown in the presence or absence of doxycycline (2 μg/mL) and Cdc7 siRNA (72 hours) or 10 μM PHA767491 (last 24 hours) as measured using the MTS assay.