Fig 1: Generation and characterization of DAO as a chemogenetic approach to study the met-abolic and transcriptomic response to intracellular H2O2 in HUVEC. (A) Lentiviral overexpression of human D-amino acid oxidase (DAO) and its chemical reaction. (B) Immuno-fluorescence for DAO in HUVEC-DAO (left) and HUVEC-CTL (empty vector, right). (C) H2O2 measurements in HUVEC using Luminol/HRP and (D) Amplex red®/HRP assay * p < 0.05 10 mM D-Ala versus 1 mM D-Ala; # p < 0.05 10 mM D-Ala plus vs 10 mM D-Ala minus PEG-catalase. One-way-ANOVA with Bonferroni correction.
Fig 2: Oxidation of peroxiredoxins (Prx) in response to exogenous or DAO-derived H2O2 or menadione. (A): Representative redox western blot (30 µg protein) for Prx1, Prx2, Prx3, and Prx-SO3 after stimulation with different concentration of D- or L-Ala in HUVEC. (B): Quantification of redox-western blots by densitometry (n = 3). (C): Redox western blot and quantification (D) for Prx1 and Prx2 with HUVEC-DAO pre-incubated with auranofin (20 min, 3 µM) prior D- or L-ala stimulation, * p < 0.05 as compared to CTL with auranofin. One-way-ANOVA with Bonferroni correction.
Fig 3: Different ROS lead to a different time-dependent transcriptomic response of HUVEC. (A): Differentially expressed genes after stimulation with 300 μM H2O2, 3 mM D-alanine or 5 μM menadione over the course of 30, 90, 270 and 900 min (significantly changed genes (padj < 0.05) are highlighted in blue and red). (B): Pathway annotation of significantly altered genes in A. (C): Venn diagram of significantly regulated genes in the treatments as indicated. (D): Correlation analysis for significantly DEGs comparing exogenous versus DAO-derived H2O2.
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