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. 2023 Apr 20;14:2271. doi: 10.1038/s41467-023-37831-z

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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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Fig. 2 — A MA plot of RNA-seq data from eWAT-derived ATMs of obese GR^flox and GR^LysMCre mice (n = 2). Red dots show differentially expressed genes (padj <0.01). B Heatmap showing gene ontology enrichment of genes with higher (left) and lower (right) expression in ATMs from GR^LysMCre compared to GR^flox mice from data shown in (A). C Macrophage polarization index based on RNA-seq counts from GR^flox and GR^LysMCre ATMs shown in (A) and pro- and anti-inflammatory ATMs from eWAT of obese mice (n = 2–3). D Heatmap depicting enrichment of up- and downregulated genes between GR^LysMCre vs. GR^flox ATMs shown in (A) and anti- vs. pro-inflammatory ATMs³³ using a one-sided hypergeometric test. E Heatmap of 2230 genes with decreased comparing GR^LysMCre vs. GR^flox ATMs from the analysis shown in (A) and increased expression pattern comparing anti- vs. pro-inflammatory ATMs³³. Macrophage polarization genes are highlighted. F SVF isolated from eWAT of obese GR^flox and GR^LysMCre mice was cultured overnight, and the supernatant was analyzed by multiplex or ELISA (INFy) (n = 5). G eWAT was stained for F4/80 by IHC, and quantified (right). Crown-like structures (CLS) were quantified (left) (n = 4, N describes individual mice). H SVF isolated from eWAT was analyzed by FACS for a fraction of pro-inflammatory (CD11c⁺;CD206⁻) and anti-inflammatory (CD11c⁻;CD206⁺) cells per F4/80 +/CD11b+ macrophages and their ratio (n = 5, N describes individual mice). I eWAT was stained for CD206 by IHC, and quantified (GR^flox n = 4, GRLysMCre n = 5, N describes individual mice). J mRNA expression of anti-inflammatory marker genes of eWAT, scWAT, and BAT was assessed by RT-qPCR (n = 5–6 eWAT, 6–7 scWAT and BAT, N describes individual mice). K eWAT was stained for CD11c by IHC and quantified (n = 5, N describes individual mice). L eWAT, scWAT, and BAT gene expression for inflammatory markers were analyzed by RT-qPCR (eWat GR^flox: Tnf n = 5, Mcp1 n = 6, Adgre1 n = 6, Il1b n = 5, Clec7a n = 6; GR^LysMCre: Tnf n = 6, Mcp1 n = 6, Adgre1 n = 6, Il1b n = 6, Clec7a n = 6. scWat GR^flox: Tnf n = 6, Mcp1 n = 6, Adgre1 n = 6, Il1b n = 6, Clec7a n = 6; GR^LysMCre: Tnf n = 6, Mcp1 n = 6, Adgre1 n = 6, Il1b n = 6, Clec7a n = 6. Bat GR^flox: Tnf n = 5, Mcp1 n = 6, Adgre1 n = 6, Il1b n = 5, Clec7a n = 6; GR^LysMCre: Tnf n = 6, Mcp1 n = 6, Adgre1 n = 5, Il1b n = 6, Clec7a n = 6, N describes individual mice). Data show mean ± SEM. Statistical analysis via two-tailed Student’s t-test and Deseq with Benjamini–Hochberg correction padj <0.01 (A); p < 0.05*, p < 0.01**, p < 0.001***, p < 0.0001****. Images were obtained at ×10 original magnification. Exact p values: G CLS: 0.0033, F4/80 Area: 0.0396, H 0.0235, I 0.0019, J eWat: Cd163: 0.0023, Arg1: 0.0830, Klf4: 0.0143, Mertk: 0.0406, scWat: Cd163: 0.0007, Mertk: 0.0493, K 0.0130, L eWat: Tnf: 0.0217, Adgre1: 0.0101, Il1b: 0.0046, Clec7a: 0.0079, scWat: Tnf: 0.0004, Adgre1: 0.0061, Clec7a: 0.0054, BAT: Tnf: 0.0410. Scale bar: 100 µm in (G, I, K).