1. EGF-induced HPSE nucleolar localization induces Topo I activity in 231BR3 cells.
EGF-induced HPSE nucleolar localization induces Topo I activity in 231BR3 cells. A, cell fractionation was done as described in Materials and Methods. The fractions were subjected to Western blotting applying anti-HPSE antibody (1453). Purity of fractions was verified using nucleoli-specific (anti-fibrillarin), nuclear-specific (anti-SC35), and cell membrane–specific (anti–integrin β1) antibodies. S, serum-free only (control); E, EGF treatment. B, a representative agarose gel showing EGF-induced Topo I activity. Nucleolar Topo I converts supercoiled DNA (Sc, provided by Topo I assay kit, TopoGen, Inc.) into a relaxed DNA form. Samples were treated with EGF (100 ng/mL) as indicated. Nucleoli were derived from (A) above. C, Topo I activity assays. Briefly, nucleoli were isolated from 231P and 231BR3 cells following treatment with or without EGF (100 ng/mL) for 30 min as per A. Topo I activity was determined by its ability to produce the relaxed DNA form, and was calculated as a ratio of relaxed/supercoiled DNA normalized against nontreated 231P. Three independent experiments were done. **, P <
Lixin Zhang et al. Mol Cancer Res 2010;8:
©2010 by American Association for Cancer Research