Figure 2
Doxorubicin-induced cell fusion. Translocation of YB-1-GFP and YB-1-Ds-Red fusion protein in respond to doxorubicin treatment. Confocal images of the two fluorescent tagged YB-1 proteins localized to each of the nuclei of non-fused MCF-7 cells and localized in one nucleus in fused MCF-7 cells. A: Control; MCF-7/YB-1-GFP transfectant without doxorubicin treatment. B--F: Demonstrate nuclear translocation of YB-1. C: Western blots of YB-1 protein from the fractionated samples; 1 × 106 cells were lysed using Pierce Nuclear and Cytoplasmic Extraction Reagent Kit. 10 μg of each cytosolic extract; CE, nuclear extract; NE proteins were analyzed by 10% SDS-PAGE and Western blotted using specific antibodies diluted 1:1000 (YB-1) or 1:10000 (GAPDH). Secondary antibody (anti-mouse) diluted 1:25,000 was used with chemiluminescent substrate for detection. Cells were grown for 6 days before visualized by phase contrast and fluorescence microscopy (Axiovert 200, Carl Zeiss). D--F: MCF-7/pcDNA3-EGFP/YB-1 and MCF-7/pDsRed2-N1/YB-1 cells were mixed (1 to 1 ratio) and co-cultured with 10 nM doxorubicin and visualized by phase contrast and fluorescence microscopy. Green; GFP, Red; dsRed, Left panels; GFP detection. Middle panels; dsRed detection. D and F: Right panels; merged (yellow or orange) images represent fused MCF-7 cells.