Fig. 4

In vitro regulation of ZBP1 down-regulation and macrophage M2 polarization by cg09897064 methylation. CD14⁺ mononuclear macrophages were transfected with the ZBP1-unmethylation overexpression plasmid (ZBP1-U), ZBP1-methylation overexpression plasmid (ZBP1-M), or blank control plasmid (Ctrl). A. Protein expression levels of Inos (M1 macrophage marker), ARG1 (M2 macrophage marker), and ZBP1 were assessed by Western blot analysis in M0,M1,and M2 macrophages. B. Flow cytometric analysis was conducted to ascertain the expression of CD86 (M1 macrophage marker) and CD206 (M2 macrophage marker) following transfection with either ZBP1-U or ZBP1-M. C-D. ELISA was used to measure the levels of IL1β (M1 macrophage marker) and IL10(M2 macrophage marker) in the culture medium of CD14⁺ mononuclear macrophages from the experimental groups. E–F. Flow cytometric analysis was performed to determine the percentage of CD68⁺CD80⁺ (M1 macrophage marker) and CD68⁺CD163⁺ (M2 macrophage marker) after transfection with either ZBP1-U or ZBP1-M. Statistical significance was established through the application of suitable tests(*P < 0.05,**P < 0.01,***P < 0.001)for the mean ± standard deviation data presented