![]() Cells were either untreated (ctrl), treated with 10 mM nicotinamide (NAM), 5 μM EX-527 or 10 μM PJ34 (top). (A) NIH3T3 cells expressing Myc-PER2 were immunostained with antibody to Myc. SIRT1 inhibitors, but not PARP1 inhibitor, regulate PER2 localization and attenuate the amplitude. The data represented as filled circle and triangle are individual data of two independent samples and bars are the average of two samples. TUBULIN was used to confirm fractionation. Each sample was normalized by LAMIN B1 protein level. PER2 protein levels were quantified using ImageJ software (bottom). (E) PER2 protein levels in nuclear extract and total cell lysate were detected under the 7.5 nM FK866-treated condition (top). ∗ p < 0.05, ∗∗ p < 0.01, compared to each subcellular localization in control cells by Student’s two-tailed t-test. The data represented are the mean ± SEM of at least three independent samples. Subcellular localizations were counted and quantified N, nucleus C, cytoplasm N + C, both nucleus and cytoplasm. The representative images were captured by a confocal laser scanning microscope. NIH3T3 cells expressing Flag-CRY1 and either Myc-PER2-WT, Myc-PER2-NLS, or Myc-PER2-NES were immunostained with antibody to Flag (D). Cells were either untreated (ctrl) or treated with 7.5 nM FK866 (FK) or 7.5 nM FK866 and 1 mM NMN (FK + NMN). NIH3T3 cells expressing Myc-PER2 (A), Myc-CRY1-Flag (B), or Myc-PER2/Flag-CRY1 (C) were immunostained with antibody to Myc or Flag (top). ![]() ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, compared to each of the untreated control by Student’s two-tailed t-test.įK866 changes PER2 subcellular localization, which is abrogated by co-treatment with NMN. ![]() All data represented here, except (E) ( n = 4), are the mean ± SEM of three independent samples. Time 12 of control cells was set to 1 for each gene. (G) Circadian profiles of Bmal1 and Rev-erbα synchronized by DEX were quantified. Each gene expression level in untreated cells (ctrl) was set to 1. Each sample was normalized by the amount of 18S rRNA. (F) Circadian gene expression levels after treatment with 7.5 nM FK866 alone or 7.5 nM FK866 and 1 mM NMN were quantified by qPCR. The value of control (ctrl) was set to 1. In this study, the amplitude was defined as the height from the first peak to the first trough. One representative result is shown for each condition. (D) Bmal1-luc oscillation patterns in NIH3T3 cells treated with FK866 with or without NMN were monitored by using a real-time luciferase monitoring system. NAD + amount was normalized by protein amount. (C) NAD + amount after 24 h treatment of 7.5 nM FK866 with or without 1 mM NMN was measured by HPLC. (B) NAD + amount after 24 h treatment of FK866 at indicated concentrations was measured by HPLC. NAM, nicotinamide NMN, nicotinamide mononucleotide NAMPT, nicotinamide phosphoribosyltransferase NMNAT1-3, nicotinamide mononucleotide adenylyltransferase 1-3. NAD+ PER2 SIRT1 circadian clock subcelluar localization.Ĭopyright © 2021 Ashimori, Nakahata, Sato, Fukamizu, Matsui, Yoshitane, Fukada, Shinohara and Bessho.įK866 changes the expression levels of circadian genes, which is abrogated by co-treatment with NMN. Thus, we anticipate that the altered PER2 subcellular localization by low NAD + is one of the complex changes that occurs in the aged circadian clock. We further found that NAD +-dependent deacetylase SIRT1 is the regulator of PER2 subcellular localization. We found that, among the core clock proteins, PER2 is mainly affected in its subcellular localization by NAD + amount, and a higher cytoplasmic PER2 localization was observed under low NAD + condition. ![]() Here, we show that low NAD + in cultured cells promotes PER2 to be retained in the cytoplasm through the NAD +/SIRT1 axis, which leads to the attenuated amplitude of Bmal1 promoter-driven luciferase oscillation. However, how low NAD + condition in cells, which mimics aged or pathophysiological conditions, affects the circadian clock is largely unknown. The amount of many metabolites in the cells/body is altered with the aging process, and the most prominent metabolite among them is the oxidized form of nicotinamide adenine dinucleotide (NAD +), which is associated with posttranslational modifications of acetylation and poly-ADP-ribosylation status of circadian clock proteins and decreases with aging. The circadian clock possesses robust systems to maintain the rhythm approximately 24 h, from cellular to organismal levels, whereas aging is known to be one of the risk factors linked to the alternation of circadian physiology and behavior.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |