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. Author manuscript; available in PMC: 2013 Sep 19.
Published in final edited form as: Biochim Biophys Acta. 2012 May 10;1823(8):1306–1315. doi: 10.1016/j.bbamcr.2012.05.003

Fig. 3. Effect of cyclin B1/Cdc2 knockdown on the development of 2ME2-induced prometaphase arrest.

Fig. 3

(A) MCF-7 cells were treated with 2 μM 2ME2 for 12 h and then immunostained with anti-cyclin B1 or anti-Cdc2 antibodies. Nuclear accumulation of cyclin B1 (left panel) and Cdc2 (right panel) in 2ME2-treated cells that have developed prometaphase arrest. (B) Subcellular localization of cyclin B1 and Cdc2 in MCF-7 cells during 2ME2-induced prometaphase arrest (at 6, 12, and 24 h after treatment). Cytosolic and nuclear extracts were prepared from 2ME2-treated cells, and cyclin B1 and Cdc2 proteins were analyzed by Western blotting. (C, D, and E) MCF-7 cells were transfected with sicyclin B1 and siCdc2 or siCon, and then treated with 2ME2 for 12 h. Total cell lysates were analyzed by Western blotting for cyclin B1 and Cdc2. The DNA content of the cells was analyzed using flow cytometry (D), and their gross nuclear morphology was examined under fluorescence microscopy (original magnification, × 200) after the cells were stained with Hoechst-33342 (E, left panel). The quantitative data for the prometaphase-arrested cells (based on counting ≥ 200 nuclei in each treatment group) are shown in E(right panel). Each bar is the mean ± S.D. from triplicate measurements. * P < 0.05 versus vehicle control; #P < 0.05 versus 2ME2 treatment.