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. 2019 Feb 7;10:633. doi: 10.1038/s41467-019-08328-5

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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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Fig. 1 — RGM-A controls macrophage phenotype and regulates human PMN/macrophage chemotaxis and chemokinesis. To mark the differentiation into the classically activated (M1) or alternatively activated (M2) phenotype, human PBMCs were stimulated with GM-CSF, M-CSF or RGM-A for 7 d, and a RGM-A transcript expression in differentiated M1 and M2 macrophages (MΦ) was quantified by RT-PCR (n = 12). b The cell morphology was analyzed by phase contrast images and measurements of the cell shape, cell length and perimeter in each high-power field (magnification ×200), Scale bar: 20 µm (n = 220). c The expression of key genes that contribute to the M1 differentiation, STAT-1 and CD80, and central genes of the M2 differentiation, Arg1 and CD163, were analyzed (n = 12). d To investigate the role of RGM-A in the phenotypic polarization of macrophages, M1 macrophages were challenged with RGM-A and subsequently with TNF-α or vehicle for 24 h. The gene expression of M1 polarization markers including STAT-1, CD40, CD80, IL-1β, and IL-6 as well as key genes of the M2 polarization such as Arg1, CD163, CD206, and IL-10 were quantified by RT-PCR (n = 14). e Schematic model of the microfluidic migration chamber. To investigate the effect of RGM-A on the regulation of e neutrophil chemotaxis (n = 10) and f, g MΦ chemotaxis/chemokinesis, chemoattractive gradients, such as N-formylmethionyl-leucyl-phenylalanine (fMLP), monocyte chemotactic protein (MCP-1) and RGM-A, were established between a range of eight peripheral wells and a central cell loading well. PMN and M1 MΦ chemotaxis were evaluated using a Casy TT cell counter (Omni Life Science, Bremen, Germany) (n = 18). f Images represent the trafficking M1 MΦ (×100 magnification). g To determine the impact of RGM-A on macrophage chemokinesis, M1 MΦ were treated with RGM-A for 10 h and the migration toward the defined gradients was evaluated using Casy TT cell counter. h The rate of MΦ clearance of human apoptotic PMNs was assessed photometrically (n = 11) and by immunofluorescence (Scale bar: 20 µm). i mRNA expression of the ALX/FPR2 and GPR32 receptors (n = 15). The results are representative of 3–8 independent experiments and are expressed as the median ± 95% CI, one-way ANOVA with Bonferroni correction (b–i), unpaired two-tailed Student’s t-test (a), *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001