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. 2020 Jul 15;48(14):7944–7957. doi: 10.1093/nar/gkaa601

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — SGT1 regulates BMAL1 stability in a necdin-dependent manner. (A) The representative immunoblots (top) and statistics data of three independent experiments (bottom) from U2OS cells transfected with control siRNA (NC) or Sgt1 siRNAs. Data are presented as means ± s.e.m.; **P < 0.01, post hoc Dunnett's t-test, one-way ANOVA. (B) Representative immunoblots (top) and statistics data of three independent experiments (bottom) from U2OS cells transfected with control siRNA (NC) or Sgt1 siRNA and treated with 10 mg/ml CHX for 0–12 h. Data are presented as means ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.005, Bonferroni post hoc analysis, two-way ANOVA. (C) The ubiquitination of BMAL1 in U2OS cells transfected with control siRNA (NC) or Sgt1 siRNA. (D) Representative immunoblots (top) and statistics data of three independent experiments (bottom) from U2OS cells transfected with different doses of NDN or empty vector (EV). Data are presented as means ± s.e.m.; *P < 0.05, post hoc Dunnett's t-test, one-way ANOVA. (E) SGT1 failed to increase the BMAL1 protein level in necdin-depleted (siNDN) U2OS cells. Representative immunoblots are shown at left; statistics data of three independent experiments are shown at right. Data are presented as means ± s.e.m.; *P < 0.05, unpaired t-test. (F) SGT1 failed to increase BMAL1 protein levels in necdin-KO MEF cells. Representative immunoblots are shown at left; statistics data of three independent experiments are shown at right. Data are presented as means ± s.e.m.; *P < 0.05, unpaired t-test. (G, H) Quantitative PCR of the indicated clock genes in U2OS cells transfected with empty vector (EV) or SGT (G), or control siRNA (NC) or NDN siRNA (H). Data are presented as means ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.005, Student's t-test, n = 3. (I) Representative bioluminescence signals from control (NC) or Sgt1-depleted (siSgt1) U2OS cells stably expressing luciferase under the control of the Bmal1 promoter. Cells were synchronized with 100 nM dexamethasone and luminescence was recorded by a Luminometer.