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. 2024 Mar 28;15:2725. doi: 10.1038/s41467-024-47008-x

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Schematic representation of RPE1^hTERT cells expressing H2B-mScarlet-I-DAAO or TOM20-mScarlet-I-DAAO (further described as DAAO^H2B and DAAO^TOM20 respectively) to induce localized intracellular H₂O₂ production. b Immunofluorescence confocal images of cell lines depicted in (a) showing colocalization of DAAO^H2B and DNA (Hoechst), and colocalization of DAAO^TOM20 with mitochondria (MitoTracker Green). Scale bar = 20 μm. c Oxygen consumption rate (OCR) upon D-Ala administration as a readout for enzymatic activity of ectopically expressed DAAO. The increase in OCR shows that both lines produce roughly similar amounts of H₂O₂ upon addition of D-Ala. Data is normalized to OCR upon Oligomycin addition, at the last timepoint before D-Ala injection. The experiment was repeated at least 3 times with reproducible results, 1 biological repeat is depicted. Data are presented as mean values of 5 separate wells -/+ SD. Source data are provided in the Source Data file. d Normalized OCR values from (c) plotted versus [D-ala]. The 6th timepoint after each D-ala injection was used for this graph. Data are presented as mean values -/+ SD. Source data are provided in the Source Data file.