(A) A schematic diagram showing the generation of endothelial cell–specific (EC-specific) Fto-deficient (EC Fto^Δ/Δ) mice. (B) Immunofluorescence of FTO protein (red), cell nuclei (DAPI, blue), and retinal microvascular ECs (CD31, green) in EC Fto^fl/fl and EC Fto^Δ/Δ mice (scale bar: 5 μm). (C) PCR genotyping verified Fto exon 3 deletion in primary retinal microvascular ECs from EC Fto^Δ/Δ mice. (D) Depletion of FTO protein in the primary retinal microvascular ECs from EC Fto^Δ/Δ mice (n = 3). (E) Dot blot showing increased m⁶A content in EC Fto^Δ/Δ mice (n = 6, Student’s t test). MB, methylene blue staining. (F) Endothelial knockout of Fto alleviated diabetes-induced retinal endothelium vascular leakage as shown in flat-mounted retinas stained with Evans blue dye. A representative image with the quantification of the fluorescence signal is shown (n = 4, scale bar: 1 mm). (G) EC Fto^Δ/Δ mice presented with fewer acellular retinal capillaries after the induction of diabetes, as indicated by retinal trypsin digestion. Red arrows indicate acellular capillaries. Acellular capillaries were quantified in 20 high-power fields and averaged (n = 4, scale bar: 50 μm). (H) Transwell assays showing that FTO enhances the migration ability of human retinal microvascular ECs (HRMECs) cultivated in high glucose. The number of migrated cells was quantified (n = 4, scale bar: 200 μm). (I) FTO increased tube formation of HRMECs treated with high glucose. The average number of tube formation for each field was assessed (n = 4, scale bar: 200 μm). NG, normal glucose (5.5 mM) with D-mannitol as osmotic control; HG, high glucose (30 mM). For F–I, significant differences were determined by 1-way ANOVA or Kruskal-Wallis’s test followed by Bonferroni’s post hoc comparison test. Data are shown as the mean ± SD. *P < 0.05.