CPT and HCPT treatment induced Topo1 protein down-regulation.

<p>IOZCAS-Spex-II cells pretreated with 0.1% DMSO (A), or 10 µM CPT (B) or HCPT (C) were harvested and lysed at different time points post treatment (0, 2, 4, 6, 12, 24 and 48 h). The cytosolic and nuclear fractions of cells were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056458#s4" target="_blank">Materials and Methods</a>. Equal amounts of protein from each fraction (40 µg for nuclear fraction and 80 µg for cytosolic fraction) were subject to electrophoresis in denaturing gels, and immunoblotted with indicated antibodies. Beta-actin was used as a loading control. The protein bands were densitometrically quantified with Quantity One (Gel Doc XR, Bio-Rad, USA). The corrected Topo1 protein was calculated as the ratio of nuclear (gray) or cytosolic (dark) fraction over beta-actin. Each bar represents the mean ±SD.