Fig. 3: Gene regulatory network uncovers FOXO3A at the hub of aging regulation.

a Heatmap showing the differentially expressed genes (O/Y DEGs, Bonferroni-corrected p value ≤ 0.05 and log₂ [fold change] ≥ 0.5) for each cell type from young and old monkeys. P values were determined by two-sided Wilcoxon rank sum tests. Rows represent genes and columns are cell types. Red, upregulated genes; blue, downregulated genes; gray, genes not differentially expressed. The number of O/Y DEGs in at least two cell populations is shown in the gray rectangles; the number of O/Y DEGs uniquely differentially expressed in each cell type is shown in the colored rectangles. See Fig. 1 for cell-type abbreviations. b GO analysis of differentially expressed genes. Red, upregulated pathways; blue, downregulated pathways. c Venn diagrams of upregulated and downregulated genes shared in AA_EC/SMC and CA_EC1/SMC of old monkeys. d Network visualization of potential transcriptional regulation in the vessels. Node size is positively correlated with the number of directed edges. Nodes and edges that directly bind to the node of FOXO3A are highlighted in red; others are gray. Red is positively correlated with the number of motif-binding sites (darker red reflects more motifs). Dashed square, enlarged area. e Violin plots with box plots inside showing FOXO3A expression across cell types. f Volcano plot of O/Y DEGs (p value ≤ 0.05, two-sided Student’s t test) verified by qPCR. Blue dots, downregulated genes; red dots, upregulated genes. g Left, immunofluorescence staining for FOXO3A and CD31 in the aortic artery. Right, quantification of immunofluorescence intensity in young and old vessel samples. Lu lumen, EC endothelial cells. Scale bar, 10 μm. Data are presented the means ± SEM; n = 8 monkeys; p values were determined by two-sided Student’s t test. h Western blotting and band intensity quantification of FOXO3 protein levels in young and old vessel samples.