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. 2017 Feb 20;7:42853. doi: 10.1038/srep42853

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Copyright © 2017, The Author(s)

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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(a) Illustration depicts YFP-DDX6 FL, NTD, and CTD. NTD: the 1–300 AA; CTD: 301–483 AA. (b) YFP-DDX6 CTD localizes to the nuclei of human cells. HeLa and HEK293T cells expressing YFP-DDX6 NTD or YFP-DDX6 CTD were imaged with fluorescent microscope. (c) Illustration depicts the YFP-tagged chimeric proteins used for nuclear localization assay. PKM, a non-shuttling cytoplasmic enzyme, was used as the cargo for nuclear transport. ; DBR1 CTD, the CTD of a splicing factor DBR1, harbors a defined K/R-rich NLS, and was used as a positive control. DBR1 CTD and DDX6 CTD were fused to the C-terminus of YFP-PKM. (d) YFP-PKM-DDX6 CTD can localize to the nuclei. HeLa cells expressing YFP-PKM, YFP-PKM-DBR1-CTD, or YFP-PKM-DDX6 CTD were imaged 2 days after transfection. A phenotypic continuum of different nucleocytoplasmic distribution exists in the cell population expressing YFP-PKM-DDX6 CTD, and was roughly categorized into 3 types. Type 1: cytoplasm-dominant; type 2: equal cytoplasm/nucleus (indicated by white arrows); type 3: nucleus-dominant. Numbers represent means and standard deviations of the percentage of each type calculated from 3 replicates. (e) The diagram showing the truncation mutants of DDX6 CTD. DDX6 CTD was further truncated into CTD-Δ1 (301–420) and CTD-Δ2 (421–483) segments. White box represents DDX6 sequence. (f) Deletions in DDX6 CTD abolish the nuclear localization of DDX6. HeLa cells expressing YFP-DDX6 CTD, YFP-DDX6 CTD-Δ1 and YFP-DDX6 CTD-Δ2 were imaged with fluorescent microscope. (b,d and f) YFP signal is shown in green. Nuclei stained with Hoechst is shown in blue. Scale bar = 20 μm.