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. 2022 Mar 2;12:3449. doi: 10.1038/s41598-022-07379-x

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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NRDC promotes the protein degradation of UCP1. (A) Time schedule of doxycycline (Doxy)-induced NRDC expression in Nrdc^−/−; Tet-on-NRDC cells, adipocytic differentiation, and the cycloheximide chase assay. Black arrows show the time points for sample collection after the treatment with cycloheximide and MG132. (B) Pictures of Nrdc^−/−; Tet-on-NRDC cells after adipocytic differentiation with (right) or without (left) Doxy-induced NRDC expression. Scale bar, 100 µm. (C) Relative mRNA levels of Nrdc, Pparg2 and Fabp4 in Nrdc^−/−; Tet-on-NRDC cells after adipocytic differentiation with or without Doxy-induced NRDC expression. Results were standardized with actb mRNA levels (in arbitrary units) and shown as the mean + SEM. N = 5. (D) Representative immunoblot image of the cycloheximide chase assay (Left panel). Signals in the immunoblot are quantified by densitometry and shown as a percentage relative to the value at time point 0 (Right panel). Data are shown as the mean (%) ± SEM of 4 independent experiments, *p < 0.05. (E) Representative immunoblot image of the cycloheximide chase assay with or without the MG132 treatment (described in A; Left panel). Signals in the immunoblot are quantified by densitometry and shown as a percentage relative to the value at timepoint 0 (Right panel). Data are shown as the mean (%) ± SEM of 4 independent experiments, *p < 0.05. (F) Time schedule of the adipocytic differentiation of Nrdc^−/−; Tet-on-NRDC cells and Doxy-induced NRDC expression with or without the MG132 treatment. Black arrows show the time points of sample collection. (G) Representative immunoblot image of the analysis described in (F) (Left panel). Signals in the immunoblot are quantified by densitometry and shown as a percentage relative to the value on Day 0 (Right panel). Data are shown as the mean (%) ± SEM of 3 independent experiments, *p < 0.05.