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. 2022 Feb 9;13:762. doi: 10.1038/s41467-022-28454-x

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© The Author(s) 2022

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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Fig. 2 — A Representative images of three independent experiments from FISH analyses using probes specific for RsmY. hMDM cells incubated with purified DiD-labelled Lp-EVs. Internalized, DiD-labelled Lp-EVs (white) 5 h post-infection. Blue, nucleus fluorescently stained using Hoechst 33342. B Human bone osteosarcoma epithelial cells stably expressing Sec61β-GFP for ER labelling (U2OS- Secβ61β cells; first three panels) and human monocyte-derived macrophages (hMDM) (right panel) were infected with DiD-labelled L. pneumophila grown until post-exponential phase (OD4.2). Images of living infected U2OS- Secβ61β cells after 1, 4, and 7 h of infection at 37 °C (5% CO₂) taken with an automated confocal microscope (Opera) are shown. Upper panel shows the cells with the green florescence labelling of the ER, lower panel shows the same cells without the green channel. Representative images of three independent experiments. White: DiD-labelled L. pneumophila and DiD-labelled Lp-EVs shed during infection; Green: ER; Blue: Nucleus (Hoechst 33342). Right panel shows fixed hMDM cells, 5 h post-infection, analyzed by confocal microscopy. White: L. pneumophila and Lp-EVs; Blue: Nucleus (Hoechst 33342).