Fig 1: Per2 binds Wee1 and Cdk1 to likely regulate their expression.(A) ChIP-qPCR analysis of P7 hearts using Per2 antibody (Ab) showing increased enrichment of two regions within the Wee1 promoter in comparison to GAPDH control. IgG, immunoglobulin G. (B) Luciferase expression assay in human stem cell–derived cardiomyocytes. Intact Wee1 promoter resulted in increased luciferase activity. Deletion of either Per2 binding sites reduced luciferase activity, and the distal Per2 binding site had a more pronounced effect. NC, negative control. (C) Schematic representation of our working model.
Fig 2: Characterization of Per2-deficient mice. (A) Schematic diagram showing target site (exons 4-6) deleted for establishment of Per2 Del4-6 mice. (B) PCR genotyping using mouse tails from wild-type (WT), heterozygous and homozygous (Per2 Del4-6 ) mice. (C) qPCR analyses of Per2 in Per2 Del4-6 and WT mice. The top panel shows the target sites of primer sets 1 and 2. (D) Western blotting analyses of PER2 protein in Per2 Del4-6 and WT mice. Western blot strips (a target protein and a loading control) were cut from one gel. Data are mean ± SD (n = 3). *p < 0.05 (t test).
Fig 3: Selective PER Induction Requires Coincident Intracellular Signals, Related to Figure 5(A) Kinase inhibition profile for BMS-754807 across a panel of protein kinases at 1 µM test concentration. Results are expressed as average percentage inhibition. Red bars indicate greater than 75% inhibition, orange greater than 50% and yellow greater than 25%. (B) Addition of insulin (600 nM) in the absence of extracellular glucose elicits a clear induction of PER2::LUC, which is potentiated by the presence of glucose extracellularly (n = 4, mean ± SEM). See Figure 5D for quantification. (C,D) Inhibition of PI3K (ZSTK474) abolishes both the acute induction of PER2 following insulin and (E) the subsequent shift in circadian phase (n = 4, mean ± SEM, 2-way ANOVA, Tukey’s multiple comparisons test). Please note that the effect of LY294002 on phase cannot be analyzed, since it abolishes PER2::LUC expression within 24 h of application. (F,G) Inhibition of mTOR with torin1 significantly attenuates the phase shift evoked by insulin (n = 4, mean ± SEM, 2-way ANOVA, Tukey’s multiple comparisons test). (H) mTOR activator MHY1485 does not induce PER2 expression comparably to insulin (n = 4, mean ± SEM), and nor does (I) simultaneous application of MHY1485 and PTEN inhibitor VO-OHpic (n = 4, mean ± SEM). (J) qPCR analysis shows levels of miR24-3p, miRNA29a-1 and miR30a-5p are all significantly reduced in PER2::LUC fibroblasts after 60 min of insulin treatment (n = 4, mean ± SEM, Welch’s t test). (K) Simultaneous inhibition of miRs 24-3p, 29a-1 and 30a-5p, pharmacological inhibition of PTEN and activation of mTOR in PER2::LUC fibroblasts recapitulates the PER2 induction by insulin, an effect not seen with any of these treatments alone or in dual combination (n = 4, representative). Extended from Figure 5I. (L,M) miRNA inhibition combined with PTEN inhibition and mTOR activation recapitulates the PER2 induction by insulin in PER2::LUC cardiomyocytes (n = 4, mean ± SEM, 2-way ANOVA, Tukey’s multiple comparisons test). (N) In fibroblasts, silencing of only miR 24-3p and mIR 30a-5p, but not mIR 29a-1, combined with inhibition of PTEN and activation of mTOR does not induce PER2::LUC comparably with insulin (n = 4, mean ± SEM, 1-way ANOVA, Tukey’s multiple comparisons test).
Fig 4: The SCN Is Robust against Resetting by Insulin(A–D) Insulin added to SCN slices (A) (top) does not (B) induce PER2::LUC (n = 3, representative) or (C) alter period, although (D) amplitude is modestly increased (n = 5, t test). SCN pre-treated with tetrodotoxin (TTX) (A) (bottom) do show (B) acute PER2 induction by insulin (n = 3, representative, TWA, Tukey’s MCT). See also Figure S2.(E and F) Time-lapse analysis of TTX-treated SCN (E) shows lateral SCN (red circle) is more responsive to insulin (F) (n = 3, representative).(G) Pixel analysis of this region shows cells maintain ~24 h period following insulin with no significant increase in ~12 h periods (n = 3, TWA, Sidak MCT).(H) Broader distribution of phases; pre-insulin = 11.05 ± 0.03 h (n = 822 pixels across 3 slices), post-insulin = 9.49 ± 0.17 h, (n = 737 pixels across 3 slices), F test variance comparison of 29.8, p < 0.0001. Mean ± SEM shown where applicable.See also Figure S3.
Fig 5: In hearts with disrupted sympathetic cardiac innervation and in Per1/Per2 DKO hearts, suppression of Wee1 kinase activates the Cdk1/cyclin B1 mitosis entry complex.(A) Schematic representation of the different proteins linking the cell cycle with the circadian cycle. Wee1 is a kinase that phosphorylates and inactivates Cdk1, not allowing the Cdk1/cyclin B1 complex to induce entry into mitosis. Cdc25 is a phosphatase with the opposite effect. (B) Gene expression analysis of factors known to link Per1/Per2 and cell cycle showed that Wee1 is consistently decreased in both hearts with inhibited sympathetic innervation and Per1/Per2 DKO. Cdc25 is significantly increased in SNi hearts. (C and D) Western blot analysis confirmed decreased expression of Wee1 protein kinase and increased expression of Cdk1 and cyclin B1 proteins in hearts with disrupted sympathetic innervation and Per1/Per2 DKO. Phosphorylated Tyr15 Cdk1 was decreased in both mouse models suggestive of increased Cdk1 activation. (E) Norepinephrine treatment of wild-type NMCMs induced Wee1 gene expression; however, this effect was not observed in Per1/Per2 DKO cardiomyocytes. Student’s t test was used for two-group analysis. ANOVA test was used for multiple group comparisons. Data are presented as means ± SEM. Only P values <0.1 are reported.
Supplier Page from Abcam for Anti-PER2 antibody