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. 2023 Jul 6;5(7):1188–1203. doi: 10.1038/s42255-023-00834-7

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© The Author(s) 2023

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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Extended Data Fig. 4 — a, Dot plot of gene expression (log₂ RPKM) of previously published liver macrophage markers (top DEG for each cluster) by Ramachandran et al. Colour intensity indicates expression level and dot size indicates gene expression frequency (percentage of cells expressing the gene). b, Dot plot of previously published myeloid cell markers (top DEG for each cluster) by Guilliams et al. c, Violin plot of gene expression distribution (log₂ RPKM; colours indicate mean expression) of signature markers used to define each LM cell population by flow cytometry analysis or sorting. Myeloid cells were defined as HLA-DR⁺CD68⁺ and LM1 was defined as CD14⁺CD206⁺CD16⁺S100A9^low, LM2 was defined as CD14⁺CD206⁺CD16^-S100A9^low, LM3 was defined as CD14⁺CD206^-CD16⁺S100A9^high, LM4 was defined as CD14⁺CD206^-CD16^-S100A9^high and cDC2 was defined as CD14^-CD206⁺CD16^-S100A9^low. d, Correlation between RNA (log₂ RPKM) and protein expression (log₂ MFI; from indexed data) of selected signature markers on single LMs (HLA-DR P<0.0001, CD4 P=0.0011, CD206 P<0.0001, CD16 P<0.0001). Data are from 5 individuals with obesity. e, Gating scheme for flow cytometry analysis of human NPCs (representative gating plots are from a lean individual) gated using FMO controls and f, proportion of human LM subset in lean individuals and individuals with obesity before (fresh, n=3) and after cryopreservation (n=3). g, Dot plot of gene expression (log₂ RPKM) of chemokines, chemokine-receptors, cytokines, M1 and M2 markers, and pattern recognition receptors in LM subsets. h, Dot plot of gene expression (log₂ RPKM) of previously published liver macrophage marker genes by MacParland et al. i, Gating scheme for sorting of each individual LM subset (LM1-LM4) from human NPCs by FACS, gated using FMO controls. Representative gating plots are from a lean individual. j, Representative images of cytospins stained with Wright-Giemsa of isolated monocytes from peripheral blood mononuclear cells from one individual. Scale bar, 10µm. Data are presented as mean ± s.e.m. P values were calculated by two-way ANOVA with adjustment for multiple comparisons (f) or by two-tailed Pearson correlation (confidence interval: 95%). *p < 0.05. RPKM, Reads per kilobase of exon model per million mapped reads; MFI; mean fluorescence intensity; ns, not significant; KC, Kupffer cells; moKCs, monocyte-derived KCs; LAMs, lipid-associated macrophages.

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