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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. 1 — A Negative staining transmission electron microscopy of Lp-EVs (n = 1). B Cryogenic transmission electron microscopy (n = 1), unilamellar EVs (black arrow), two lipid bilayer EVs (white arrow). C Fluorescence microscopy, DiD (red) and Syto-RNAselect (green) labelled Lp-EVs, (n = 5). D Absolute particle concentration (left) and median size (right) of purified Lp-EVs determined by ZetaView. Each dot represents a single measurement performed in triplicates and the (mean) SD of n = 5. Statistical analysis performed using unpaired t-test (two-tailed, p < 0.05 significant). Red Square, representative size distribution of particles for each sample. *p = 0.0112 (Wilcoxon). ns: p > 0.05 (Wilcoxon). Source data provided as Source data file. E Size distribution pre- and post-floatation was unchanged. F Absolute particle concentration (left) and median size (right) measured through light scatter (All) and fluorescence (DiD) mode. Each dot is one measurement and the (mean) SD of n = 3, ns: p > 0.05 (Wilcoxon). Source data provided as Source data file. G THP-1 cells infected for 8 h with L. pneumophila wt or ∆rsmY. Data are presented as (mean) SD of n ≥ 3 independent, biological replicates (p < 0.0001) H) THP-1 incubated 3 h with wt or ∆rsmY Lp-EVs. Data are presented as (mean) SD of n = 5 independent biological replicates. (p < 0.0001). (G + H) Protein quantities of RIG-I and IRAK analyzed by western blot, intensities relative to the non-infected control (NI) and RhoGDI loading control. A two-way ANOVA for statistical analysis was performed. Right, representative western blots. Source data provided as Source data file. I Quantification of RIG-I, IRAK1 and cRel protein levels after transfection of THP-1 with RsmY and tRNA-Phe. Relative mean intensities normalized to non-transfected cells (NT) and RhoGDI loading control. Control RNA (ctrlRNA) average of random L. pneumophila DNA, anti-sense of RsmY or tRNA-Phe. Left, representative blot. Data are presented as (mean) SD of n = 8 independent biological replicates. For statistical analysis a two-way ANOVA was performed with p < 0.05 significant (*), p < 0.01 very significant (**), p < 0.001 extremely significant (***). Source data provided as Source data file. J Representative EMSA (n = 3) of in vitro transcribed RNA.