Figure 1

14-3-3σ is involved in DNA damage -induced COP1 nuclear exclusion. (A) Live-cell imaging of NIH3T3 cells expressing RFP-COP1. Typical subcellular localization of RFP-COP1 (red, indicated by arrow) is shown. Hoechst 33342 (0.04 μg/mL) was added for DNA staining (blue). Merged image containing phase-contrast image is also indicated. Scale bar, 10 μm. (B) Dynamic cell shuttling of RFP-COP1 in NIH3T3 cells was impaired in the presence of 10 Gy ionizing radiation (IR). Images were collected at 20-min intervals. The minutes indicate the elapsed time since the first picture was taken (30 min after treatment). (C) COP1 cytoplasmic localization is regulated in response to doxorubicin and IR. U2OS cells stably expressing RFP-COP1 were treated with 10 Gy ionizing radiation (IR) or 1 μg/mL doxorubicin. Subcellular localization of RFP-COP1 is shown by Western. Lamin B1 was used as the marker of nuclear (N) fraction, while α-tubulin was used as the marker for cytoplasmic (C) fraction. (D) 14-3-3σ inhibits COP1 nuclear shuttling. RFP-COP1-expressing cells were infected with Ad-β-gal or Ad-14-3-3σ. Live-cell images were captured using a confocal microscope at the indicated time points. Arrows indicate the representative COP1 translocation from the cytoplasm to the nucleus in the Ad-β-gal treatment group and lack of nuclear migration in the Ad-14-3-3σ group. Bar graph shows the percentage increase in nuclear COP1 staining in each group. Error bars represent 95% confidence intervals. Scale bar= 10 μm. Cell fractionation of RFP-COP1 in each group is shown by Western blot. (N), nuclear fraction. (C), cytoplasmic fraction.