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. 2019 Sep 26;9:13931. doi: 10.1038/s41598-019-50329-3

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© The Author(s) 2019

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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The kinetically determined, CAP-induced processes show differential response to early and late addition of inhibitors. The experiments were performed in analogy to those described in Fig. 10. In addition, parallel assays also received either 2 mM of the singlet oxygen (¹O₂) scavenger histidine or 25 µM of the peroxynitrite (ONOO⁻) decomposition catalyst FeTPPS after the washing step that followed CAP treatment and the first incubation step of 25 min. Assays without CAP treatment showed less than 5% apoptotic cells at all time points (not shown in the graph). (A) Process #1 was inhibited when histidine was present during CAP treatment and the first 25 min incubation step, but not when histidine had been added after the washing step. Likewise, FeTPPS added after the washing step had no inhibitory effect. (Note that addition of FeTPPS during CAP treatment would have shifted process #1 to process #3, shown below). Process #1 is the overall process, based on unlimited primary and secondary singlet oxygen generation. (B) Process #2 was induced when AEBSF was present during CAP treatment and the 25 min incubation step. Process #2 was completely inhibited by the presence of histidine during CAP treatment and the 25 min incubation step, and partially inhibited when histidine or FeTPPS had been added after the washing step. Process #2 reflects the regime in which secondary ¹O₂ generation is blocked during CAP treatment and the subsequent 25 min incubation step. After the washing step, secondary ¹O₂ generation resumes, driven by the imprinted signature established initially by primary ¹O₂generated by long-lived species of CAP-treated medium. (C) Process #3 is independent of ONOO⁻ and NOX derived O₂^●− during CAP treatment and the 25 min incubation step, but completely dependent on ¹O₂ at this step. The process after the washing step is completely dependent on ¹O₂ and on ONOO⁻. Process #3 depends on the imprinted signature by primary ¹O₂ from the gaseous phase of CAP. The imprinted signature drives secondary ¹O₂ generation after the washing step.