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. 2018 Aug 29;8:12998. doi: 10.1038/s41598-018-31171-5

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© The Author(s) 2018

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 increase in TLR4 surface expression and phagocytosis of E. coli caused by Rab9a silencing is rescued by the expression of GFP-Rab9a or blocking of TLR4, respectively. (a) Macrophages were treated with siRNA for Rab9a, Rab14 or siRNA control and the levels of TLR4 analyzed by flow cytometry. Columns represent the mean fluorescence intensity (MFI) of TLR4 in macrophages treated with the indicated siRNAs. Error bars indicate the standard error of the mean of four independent experiments. Statistical significance (***p < 0.001) refers to the difference between macrophages treated with siRNA for Rab9a and macrophages treated with siRNA control. (b) Macrophages were treated with siRNA for Rab9a or siRNA control, followed by expression of GFP-tagged Rab14 or Rab9a. Cell surface levels of TLR4 of the GFP-negative and positive populations were analyzed by flow cytometry. Columns represent the mean fluorescence intensity (MFI) of TLR4 in macrophages treated with the indicated siRNAs with or without expression of GFP-tagged Rabs. Error bars represent the standard error of the mean of two independent experiments. Statistical significance (**p < 0.01) refers to the difference between macrophages treated with siRNA for Rab9a and macrophages treated with siRNA control. (c) Macrophages were treated with siRNA control or siRNA for Rab9a, incubated or not with TLR4 blocking antibody and infected with YFP-E. coli. Cells were incubated for 15 minutes, washed and chased for different time points. Cells were then analyzed by flow cytometry. Columns represent the percentage of positive cells for CD11b and YFP, analyzed after different times of incubation. Error bars indicate the standard error of the mean of four independent experiments. Statistical significance refers to the difference between macrophages treated with siRNA for Rab9a and macrophages treated with siRNA control (**p < 0.01; ***p < 0.001) and to the difference between macrophages treated with siRNA control and macrophages treated with siRNA control and TLR4 blocking antibody (^#p < 0.05; ^##p < 0.01).