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HEK293 cells express dystrophin Dp71 with nucleus-specific localization of Dp71ab

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Abstract

The dystrophin gene consists of 79 exons and encodes tissue-specific isoforms. Mutations in the dystrophin gene cause Duchenne muscular dystrophy, of which a substantial proportion of cases are complicated by non-progressive mental retardation. Abnormalities of Dp71, an isoform transcribed from a promoter in intron 62, are a suspected cause of mental retardation. However, the roles of Dp71 in human brain have not been fully elucidated. Here, we characterized dystrophin in human HEK293 cells with the neuronal lineage. Reverse transcription-PCR amplification of the full-length dystrophin transcript revealed the absence of fragments covering the 5′ part of the dystrophin cDNA. In contrast, fragments covering exons 64–79 were present. The Dp71 promoter-specific exon G1 was shown spliced to exon 63. We demonstrated that the Dp71 transcript comprised two subisoforms: one lacking exon 78 (Dp71b) and the other lacking both exons 71 and 78 (Dp71ab). Western blotting of cell lysates using an antibody against the dystrophin C-terminal region revealed two bands, corresponding to Dp71b and Dp71ab. Immunohistochemical examination with the dystrophin antibody revealed scattered punctate signals in the cytoplasm and the nucleus. Western blotting revealed one band corresponding to Dp71b in the cytoplasm and two bands corresponding to Dp71b and Dp71ab in the nucleus, with Dp71b being predominant. These results indicated that Dp71ab is a nucleus-specific subisoform. We concluded that Dp71, comprising Dp71b and Dp71ab, was expressed exclusively in HEK293 cells and that Dp71ab was specifically localized to the nucleus. Our findings suggest that Dp71ab in the nucleus contributes to the diverse functions of HEK293 cells.

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Acknowledgments

This work was supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (KAKENHI; 24390267 and 26860803), a Research Grant for Research on Psychiatric and Neurological Diseases and Mental Health from Japan Agency for Medical Research and Development, AMED, and an Intramural Research Grant for Neurological and Psychiatric Disorders from the National Center of Neurology and Psychiatry (NCNP). We are grateful for technical assistance from Mina Murakami.

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Authors and Affiliations

  1. Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, 518 Arise, Ikawadani, Nishi, Kobe, 651-2180, Japan

    Atsushi Nishida, Sato Yasuno, Emma Tabe Eko Niba & Masafumi Matsuo

  2. Kobe Pharmaceutical University, Higashinada, Kobe, 658-8558, Japan

    Sato Yasuno & Atsuko Takeuchi

  3. Department of Pediatrics, Graduate School of Medicine, Kobe University, Chuo, Kobe, 650-0017, Japan

    Hiroyuki Awano

  4. Department of Pediatrics, Hyogo College of Medicine, Mukogawacho, Nishinomiya, 663-8501, Japan

    Tomoko Lee & Yasuhiro Takeshima

  5. Department of Pathology and Applied Neurobiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, 602-8566, Japan

    Takahiro Fujimoto & Kyoko Itoh

  6. Department of Community Medicine and Social Healthcare Science, Graduate School of Medicine, Kobe University, Chuo, Kobe, 650-0017, Japan

    Hisahide Nishio

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  1. Atsushi Nishida
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  2. Sato Yasuno
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  9. Yasuhiro Takeshima
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  10. Hisahide Nishio
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Correspondence to Masafumi Matsuo.

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MM is an advisor for JCR Pharma Co., Japan, and Daiichi Sankyo Co., Ltd., Japan. The other authors declare that they have no competing interests.

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Nishida, A., Yasuno, S., Takeuchi, A. et al. HEK293 cells express dystrophin Dp71 with nucleus-specific localization of Dp71ab. Histochem Cell Biol 146, 301–309 (2016). https://doi.org/10.1007/s00418-016-1439-2

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