Fig. 3

This study examines the effects of cg09897064 methylation on ZBP1 expression and chromatin accessibility during macrophage polarization. A-D. ZBP1 protein expression: M1 and M2 macrophage polarization models were established by treating CD14⁺ mononuclear macrophages with IFNγ and IL4, respectively. An evaluation of protein expression levels of iNOS (a M1 macrophage marker), ARG1(a M2 macrophage marker), and ZBP1 in M0, M1, and M2 macrophages was conducted via Western blot analysis. The relative levels of iNOS and ARG1 proteins verified the successful polarization of macrophages;ZBP1 was highly expressed in M1 macrophages,while it was found to be low in both M0 and M2 macrophages. E–F. Employing methylation-specific PCR(MSP)and bisulfite amplicon sequencing(BSAS),the methylation status of cg09897064 in M0,M1,and M2 macrophages was analyzed. G. chromatin accessibility: ATACseq heatmap and peak chart visualization were utilized to evaluate the opening or closing of chromatin regions, specifically on ZBP1 and its promoter region, during macrophage polarization. Statistical significance was determined by conducting appropriate tests (P < 0.05 for significance: *, P < 0.01 for high significance: **, and P < 0.001 for very high significance: ***) on the mean ± standard deviation of the provided dat