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Open AccessArticle

Generation of a Quantitative Luciferase Reporter for Sox9 SUMOylation

1
Laboratory of Animal Regeneration Systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kanagawa 214-8571, Japan
2
Department of Developmental Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo, Aoba, Sendai, Miyagi 980-8575, Japan
3
Meiji University International Institute for Bio-Resource Research, Kanagawa 214-8571, Japan
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(4), 1274; https://doi.org/10.3390/ijms21041274
Received: 17 January 2020 / Revised: 30 January 2020 / Accepted: 11 February 2020 / Published: 13 February 2020
(This article belongs to the Section Molecular Biology)
Sox9 is a master transcription factor for chondrogenesis, which is essential for chondrocyte proliferation, differentiation, and maintenance. Sox9 activity is regulated by multiple layers, including post-translational modifications, such as SUMOylation. A detection method for visualizing the SUMOylation in live cells is required to fully understand the role of Sox9 SUMOylation. In this study, we generated a quantitative reporter for Sox9 SUMOylation that is based on the NanoBiT system. The simultaneous expression of Sox9 and SUMO1 constructs that are conjugated with NanoBiT fragments in HEK293T cells induced luciferase activity in SUMOylation target residue of Sox9-dependent manner. Furthermore, the reporter signal could be detected from both cell lysates and live cells. The signal level of our reporter responded to the co-expression of SUMOylation or deSUMOylation enzymes by several fold, showing dynamic potency of the reporter. The reporter was active in multiple cell types, including ATDC5 cells, which have chondrogenic potential. Finally, using this reporter, we revealed a extracellular signal conditions that can increase the amount of SUMOylated Sox9. In summary, we generated a novel reporter that was capable of quantitatively visualizing the Sox9-SUMOylation level in live cells. This reporter will be useful for understanding the dynamism of Sox9 regulation during chondrogenesis. View Full-Text
Keywords: Sox9; SUMO; NanoBiT; PTM; cartilage; chondrocyte Sox9; SUMO; NanoBiT; PTM; cartilage; chondrocyte
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MDPI and ACS Style

Saotome, H.; Ito, A.; Kubo, A.; Inui, M. Generation of a Quantitative Luciferase Reporter for Sox9 SUMOylation. Int. J. Mol. Sci. 2020, 21, 1274.

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