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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. 2 — a–d H₂O₂ measurements using the ratiometric HyPer7 sensor to determine diffusion of DAAO-produced H₂O₂. Treating cells with 400 µM H₂O₂ was used to determine 100% HyPer7 oxidation. Lowest oxidation measured was set to 0%. NES-HyPer7, localized in the cytosol, becomes already oxidized at the lowest concentration (2.5 mM) of D-Ala used in RPE1-hTERT-DAAO^TOM20 cells (a) In contrast, nuclear NLS-HyPer7 oxidation is not detected at 0–10 mM D-Ala. b NLS-HyPer7 oxidation is already detected at 2.5 mM D-Ala in RPE1-hTERT-DAAO^H2B cells (c) while NES-HyPer7 oxidation is only detected starting from 10 mM D-Ala. d Data are presented as mean values -/+ SD, n = 3 biological replicates. Source data are provided in the Source Data file. e Representative image of crystal violet staining of DAAO lines after 24 h L/D-Ala treatment. Both RPE1-hTERT-DAAO^H2B and RPE1-hTERT-DAAO^TOM20 cells do not survive continuous D-Ala treatments above 10 mM D-Ala. Experiment was repeated at least 3 times, a representative image is shown.