Fig. 4: FOXO3A^−/− ECs exhibited impaired cell proliferation and migration.

a Schematic showing the method used to generate FOXO3A^+/+ and FOXO3A^−/− heVECs. b Representative bright-field and immunostaining for FOXO3A in FOXO3A^+/+ and FOXO3A^−/− heVECs. Scale bar, 50 or 5 μm. c Upper, clonal expansion analysis of FOXO3A^+/+ and FOXO3A^−/− heVECs. Bottom, areas positive for crystal violet staining were quantified by ImageJ. n = 3 independent experiments. Scale bar, 100 µm. d The percentage of heVECs at each cell-cycle stage. n = 3 independent experiments. e In vitro angiogenesis is assessed by the formation of capillary-like tubes from FOXO3A^+/+ and FOXO3A^−/− heVECs. The red circle represents an intact tube. n = 4 independent experiments. Scale bar, 100 μm. f Left, representative wound scratch assay for detecting changes in FOXO3A^−/− heVECs migration compared with wild-type heVECs migration. Red lines represent scar boundaries. n = 3 independent experiments. Scale bar, 100 μm. Right, quantification of wound scratch assay results. g Whole-genome sequencing analysis of copy number variation (CNV) in FOXO3A^+/+ and FOXO3A^−/− heVECs. h Heatmap showing all differentially expressed genes in FOXO3A^+/+ and FOXO3A^−/− hESCs and heVECs. i Top eight GO terms shared by O/Y DEGs and FOXO3A^+/+/FOXO3A^−/− DEGs. j A diagram showing seven core FOXO3A-regulated genes, as defined by common genes between O/Y DEGs of aged monkey ECs, FOXO3A KO DEGs in FOXO3A^+/+ and FOXO3A^−/− heVECs, and FOXO3A target genes predicted by SCENIC in aged monkeys. k KEGG pathway of 64 common genes overlapped between O/Y DEGs of aged monkey ECs and FOXO3A KO DEGs of heVECs. l A working model for FOXO3A in the homeostatic regulation of aged primate vessels. Data are presented as mean ± SEM; p values were determined by two-sided Student’s t test (c–f) or corrected by the Benjamini–Hochberg algorithm (i, k).