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FTO regulates EC-pericyte crosstalk to trigger diabetes-induced microvascular dysfunction in mice. (A) Experimental scheme for (B–K). (B, C) Representative images of fundus photos (B) and FFA (C) of control, STZ mice without injection, and STZ mice intra-vitreal injected with AAV-blank/AAV-Fto at indicated time points after injection. n = 3 per group. Blue arrows represent cotton wool spot-like lesions. Red arrow indicates leakage spot. Yellow arrow suggests abnormal vascular perfusion. (D, E) Fluorescence of Texas red dextran (D) and Evans blue (E) are visualized in retinal flat mounts of control, STZ mice without injection, and STZ mice intra-vitreal injected with AAV-blank/AAV-Fto. n = 3 per group. Representative images along with quantification results of leakage area and leaky flourescence intensity are shown. Scale bar: 2000 µm ((D) and up in (E)); 50 µm (below in (E)). (F) Immunofluorescence staining of VE-cadherin and IB4 in retinal flat mounts of control, STZ mice without injection, and STZ mice intra-vitreal injected with AAV-blank/AAV-Fto. n = 6 per group. Scale bar: 50 µm (upper); 20 µm (below). (G) Immunoblotting of ZO-1 in neural retinas collected from control, STZ mice without injection, and STZ mice intra-vitreal injected with AAV-blank/AAV-Fto. Gapdh is used as internal control. n = 3 per group. (H) Immunofluorescence staining of NG2 and IB4 in retinal flat mounts of control, STZ mice without injection, and STZ mice intra-vitreal injected with AAV-blank/AAV-Fto. n = 5 per group. Yellow arrowheads indicate pericytes. Scale bar: 50 µm. (I) Immunofluorescence staining of PDGFRβ and IB4 in retinal flat mounts of control, STZ mice without injection, and STZ mice intra-vitreal injected with AAV-blank/AAV-Fto. n = 5 per group. Scale bar: 50 µm. (J) PAS staining of trypsin digested retinal vessels isolated from control, STZ mice without injection, and STZ mice intra-vitreal injected with AAV-blank/AAV-Fto. n = 14 per group. Scale bar: 50 µm. (K) Fluorescence staining of IB4 in retinal flat mounts of control, STZ mice without injection, and STZ mice intra-vitreal injected with AAV-blank/AAV-Fto. n = 3 per group. IRMAs are represented by white and black rectangles. Capillary dropout regions are suggested by yellow arrowheads. White brackets indicate structures of main vessels, and white asterisks represent activated microglia cells wrapping around retinal vessels. Scale bar: 2000 µm (up); 50 µm (below). Data information: Data represent different numbers (n) of biological replicates. Data are shown as mean ± SEM. One-way ANOVA followed by Bonferroni’s test is used. NS: not significant (p > 0.05); *p < 0.05; **p < 0.01; and ***p < 0.001. Source data are available online for this figure.
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