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. 2022 Dec 13;133(3):e161759. doi: 10.1172/JCI161759

Figure 5. LEENE RNA promotes ischemic recovery in vivo.

Figure 5

(A) Design of the rescue experiment: Adenovirus driving GFP (Ad-GFP) or LEENE (tagged by GFP) (Ad-LEENE) was injected intramuscularly on days 1 and 5 after HLI into WT or KO mice fed an HFHS diet. (B and C) Staining of CD31 and GFP (B) and smRNA FISH of LEENE and IB4 staining with DAPI counterstain (C) in the hind limb muscle of KO mice receiving Ad-LEENE. Arrows indicate colocalization of LEENE and IB4 signals. Scale bars: 50 μm. (D) qPCR of LEENE in EC-enriched and non–EC-enriched fractions isolated from the gastrocnemius muscle (n = 3–5/group). **P = 0.006 based on 1-way ANOVA followed by Tukey’s test. (E and F) Representative flowgraphy images (E) and quantitative analysis of perfusion recovery rate in the hind limbs (n = 4/group) (F). ****P < 0.0001 based on 2-way ANOVA followed by Tukey’s test. (G) IB4 staining in the hind limb muscle (n = 3–5/group). Scale bar: 100 μm. (H) PCA plot showing the gene expression of 3 groups profiled by RNA-seq with 2 replicates per group. (I) Venn diagram showing the LEENE-rescued genes, namely the overlap between downregulated by Leene KO (WT + GFP vs. KO + GFP) and upregulated by LEENE overexpression in KO (KO + GFP vs. KO + LEENE).