Fig. 4: Alterations of macrophages aggravate adipose tissue catabolic activity.

a Bar plot showing the proportion of manually annotated macrophages in all immune cells isolated from the scRNA-seq data. Bars indicate means ± SEM. P-values were determined by unpaired Student’s t-test. ***P < 0.001. b The proportion of macrophages in SVF of inguinal and eWAT in both control and C26 mouse model group (animals implanted with C26 colon adenocarcinoma cells) determined by flow cytometry. Bars indicate means ± SEM. ns, not significant. ***P < 0.001. c The scatter plot showing the positive correlation between the proportion of macrophages in eWAT and the weight of eWAT of mouse model. Linear regression and Pearson’s correlation test with 95% CI were performed and the line of best fit was shown in the plot. d Volcano plot showing the DEGs between macrophages from patients with or without cachexia in scRNA-seq data. Red dots represented the selected genes of interest with significant upregulation during CAC, whereas blue dots were downregulated genes. e, f Bar plots showing the tumor-free weight (e) and the weight of inguinal and eWAT (f) of mice in control group (animals with no tumor cell implanted), C26 group (animals implanted with C26 tumor cells and treated with control liposomes) and C26⁺ clodronate group (animals implanted with tumor cells and treated with clodronate-liposomes). Bars indicate means ± SEM. *P < 0.05. (n = 6–8/group). g Bar plot showing the concentration of serum non-esterified fatty acids. *P < 0.05. (n = 5–6/group). h Representative images of H&E staining for epididymal WAT sections. Scale bar: 50 μm. i Quantification of the area of adipocytes in three randomly selected fields from each eWAT tissue per group in H&E staining sections. The violin plot showed the density of the estimated mean diameters of each adipocyte in captured images. The inner box was bounded by the upper (75%) and lower (25%) quantiles, with the median represented by the bold horizontal line. ****P < 0.0001. j Western blot analysis of the expression of ATGL and the phosphorylation level of HSL in eWAT tissue (n = 3/group). k Quantification of the phosphorylation level of HSL from the results of panel j. *P < 0.05. Values are means ± SEM. l qPCR analyses showing the relative mRNA levels of IFNG, TNF, and IL6 in eWAT tissue (n = 4–5/group). **P < 0.01. All P-values were determined by one-way ANOVA with post hoc Dunnett’s multiple-comparison correction tests. m Western blot analysis of IFN-γ in eWAT tissue (n = 4/group).