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. 2019 Oct 1;9:14050. doi: 10.1038/s41598-019-50320-y

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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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Assessment of autophagic activity and mTORC1 signaling in macrophage polarization during infection with Ehrlichia. Assessment of autophagic activity in bone marrow-derived macrophages (BMM) infected with E. muris or IOE was carried out by western blot analysis. BMM were incubated or not with bacteria for 24 h prior to obtaining protein extracts from the different studied groups (E. muris-, IOE-infected and uninfected BMM). Flow cytometry was used to assess the impact of mTORC1 signaling in macrophage polarization via stimulation of BMM with mTORC1 inhibitor, rapamycin (Rapa) 10 μM, by the time of infection. (A) Measurement of LC3I and LC3II in the protein extracts accompanied by quantitative analysis of LC3II/LC3I ratio of conversion. Ratio of conversion was assessed after normalization of densitometry values based on the levels of β-actin. Levels of p62 and downstream targets of mTORC1 signaling, pS6 and p4E-BP1, in the considered protein extracts are also shown. Quantitative analyses expressed as relative densitometry are shown for each marker. Full-length blots are presented in Supplementary Fig. S6, from which data were cropped and assembled. (B) Flow cytometry dot plot showing region of considered BMM (CD11b⁺ F4/80⁺ cells gated on live cells) in which the percentage of MHC class II^high-expressing cells and the mean fluorescence intensity (MFI) of surface CD206 were measured. Values near the regions represent percentage and numbers above histograms indicate MFI values. (C) Quantitative analysis for flow cytometric determinations. All quantitative analyses are exhibited as mean and standard deviation. Asterisks represent relevant statistical difference between groups. Data is representative of at least two independent experiments.