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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. 5 — A Quantitative analyses of the protein and phosphorylation levels of key proteins of the RLR and TLR signalling pathway analyzed after 3 h of-incubation of THP-1 cells with wt or the ΔrsmY Lp-EVs. wt (grey), ∆rsmY (blue). Mean intensities of the proteins, normalized against the value of non-infected cells and the RhoGDI loading control. Bars show the (mean) SD of n = 4 independent experiments. For statistical analysis, a two-way ANOVA with values of p < 0.05 significant (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****) was performed. Source data provided as Source data file. B Extracellular IFN-β concentration of THP-1 cells incubated for 3 h with wt or ΔrsmY Lp-EVs. The bars indicate the (mean) SD of n = 10 independent experiments using an unpaired t-test for statistical analysis with two-tailed values of of p < 0.05 significant (p = 0.0002). C THP-1 cells were transfected with in vitro transcribed RsmY, tRNA-Phe RNA, or non-specific control RNA (ctrlRNA). The value for the ctrlRNA comprises the results of two control RNAs, a random short L. pneumophila DNA and the anti-sense sequences of RsmY (as-RsmY). Bars show the (mean) SD of n = 3 independent experiments using an unpaired t-test for statistical analysis (two-tailed), p < 0.05 significant (p = 0.0007). In B and C the IFN-ß concentration of the supernatant was quantified and normalized against the values of the corresponding non-infected/non-transfected experiments. D THP-1 cells were transfected with siRNA targeting Rig-I (ddx58), IRAK1 (irak1), or a scramble control (neg ctrl). The protein expressions of RIG-I and IRAK1 were analyzed at time point T0, before infection with L. pneumophila. The graph shows the percentage of colony forming units (% cfu) at 24, 48, and 72 h post-infection relative to the scramble siRNA (neg ctrl). Bars show the (mean) SD of at least n = 7 independent experiments. For statistical analysis, a two-way ANOVA with values of p < 0.05 significant (*), p < 0.01 very significant (**), p < 0.001 extremely significant (***) and p < 0.0001 (****) was performed. Source data provided as Source data file.