Figure 6.

mTOR signalling is inhibited in Trx1cKO mice. (A) Enhanced oxidation of mTOR in Trx1cKO mice. Oxidation of mTOR was examined by SDS-PAGE under non-reducing conditions and subsequent western blot analysis. (Top) Cardiomyocytes were treated with 100 μM H₂O₂ for 30 min and shTrx1 for 7 days. The H₂O₂-induced mTOR band shift was abolished with 2ME. (Bottom) The band shift of mTOR in Trx1cKO mice. Ratio of oxidized (higher molecular weight) and reduced form of mTOR band intensity is indicated. (B) Inhibition of mTOR signalling in Trx1cKO mice. The phosphorylation status of mTOR substrates such as S6K and 4EBP1 was examined. (C) Schematic representation of reporter gene constructs. TSS, transcription start site; UTR, untranslated region. (D) Reporter gene assays were performed with luciferase reporter genes driven by the endogenous promoter sequences of the indicated genes. (E) Trx1 knockdown inhibits mitochondrial respiration. Oxygen consumption was measured in cardiomyocytes treated with shTrx1. Basal, ATP-production-coupled and maximum oxygen consumption rates were measured. (F) Autophagy is not significantly altered in homozygous Trx1cKO mice. The level of LC3-II and p62 was evaluated with immunoblot analyses. N=5 (A), 6–7 (B), 6–8 (D) 8 (E, upper), 24 (E, lower), and 7 (F). Statistical significance was determined with Mann–Whitney U test (D, Ndufa1 and F, p62/Tubulin). Besides this, statistical significance was determined with the Student’s t test. *P<0.05, **P<0.01, and ***P<0.001.