Selective PER Induction Requires Coincident Intracellular Signals, Related to
(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 D 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 I. (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).