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    Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels
    (Elsevier B.V., 2022) Sevimli, Gülşah; Smith, Matthew J.; Akgül Çağlar, Tuba; Bilir, Şükriye; Seçilmiş, Melike; Altun, Hamza Y.; Yiğit, Esra N.; Yang, Fan; Keeley, Thomas P.; Malli, Roland; Öztürk, Gürkan; Mann, Giovanni E.; Eroğlu, Emrah
    Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study exploits the metalloprotein geNOps in cultured HEK293T epithelial and EA.hy926 endothelial cells to test the iron-dependency in cells adapted to standard room air (18 kPa O2) or physiological normoxia (5 kPa O2). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate for the first time that cells adapted to physiological normoxia require significantly lower iron compared to cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality.
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    Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels (vol 53, 102319, 2022)
    (Elsevier, 2022) Sevimli, Gülşah; Smith, Matthew J.; Akgül Çağlar, Tuba; Bilir, Şükriye; Seçilmiş, Melike; Altun, Hamza Y.; Yiğit, Esra N.; Yang, Fan; Keeley, Thomas P.; Malli, Roland; Öztürk, Gürkan; Mann, Giovanni E.; Eroğlu, Emrah
    The authors regret that the cited reference list in the online PDF of this article contains a duplicate reference (Ref 34 and incomplete Ref 56). Ref 56 should read as follows.
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    Redox and lipidomic analysis of human brain microvascular endothelial cells (hcmec/d3) adapted to physiological oxygen levels
    (Elsevier Science Inc, 2024) Yang, Fan; Simon, Daniel; Eroğlu, Emrah; Aarsland, Dag; Siow, Richard C. M.; Mann, Giovanni E.
    The majority of studies with endothelial and other cell types are conducted in standard culture incubators gassed with 5% CO2 and room air (18 kPa O2), well known to induce oxidative stress.
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    Visualizing hydrogen peroxide and nitric oxide dynamics in endothelial cells using multispectral imaging under controlled oxygen conditions
    (2024) Altun, Hamza Yusuf; Seçilmiş, Melike; Yang, Fan; Akgül Çağlar, Tuba; Vatandaşlar, Emre; Toy, Muhammed Fatih; Vilain, Sven; Mann, Giovanni E.; Öztürk, Gürkan; Eroğlu, Emrah
    The complex interplay between hydrogen peroxide (H2O2) and nitric oxide (NO) in endothelial cells presents challenges due to technical limitations in simultaneous measurement, hindering the elucidation of their direct relationship. Previous studies have yielded conflicting findings regarding the impact of H2O2 on NO production. To address this problem, we employed genetically encoded biosensors, HyPer7 for H2O2 and geNOps for NO, allowing simultaneous imaging in single endothelial cells. Optimization strategies were implemented to enhance biosensor performance, including camera binning, temperature regulation, and environmental adjustments to mimic physiological normoxia. Our results demonstrate that under ambient oxygen conditions, H2O2 exhibited no significant influence on NO production. Subsequent exploration under physiological normoxia (5 kPa O2) revealed distinct oxidative stress levels characterized by reduced basal HyPer7 signals, enhanced H2O2 scavenging kinetics, and altered responses to pharmacological treatment. Investigation of the relationship between H2O2 and NO under varying oxygen conditions revealed a lack of NO response to H2O2 under hyperoxia (18 kPa O2) but a modest NO response under physiological normoxia (5 kPa O2). Importantly, the NO response was attenuated by L-NAME, suggesting activation of eNOS by endogenous H2O2 generation upon auranofin treatment. Our study highlights the intricate interplay between H2O2 and NO within the endothelial EA.hy926 cell line, emphasizing the necessity for additional research within physiological contexts due to differential response observed under physiological normoxia (5 kPa O2). This further investigation is essential for a comprehensive understanding of the H2O2 and NO signaling considering the physiological effects of ambient O2 levels involved.