Fig 2: CKD serum induced endothelial dysfunction and ANRIL upregulation.A Protein and mRNA levels of vWF, eNOS, VCAM-1 in HUVECs exposed to CKD serum. B Protein levels of BDNF in HUVECs exposed to CKD serum. C Representative immunofluorescence images of VCAM-1 in HUVECs exposed to CKD serum. Scale bars = 50 µm. D Mitosox Red staining showed the mitochondrial ROS level of endothelial cells. Scale bars = 50 µm. E Mitotracker Red probe was used to label mitochondria. Scale bars = 20 µm. F Representative immunofluorescence images of Mfn2 in HUVECs exposed to CKD serum. Scale bars = 50µm. G Expression of Mfn2, Drp1 in HUVECs were detected by Western blot. H ANRIL expression in HUVECs were detected by realtime PCR. I FISH assays was used to detect the expression and subcellular location of ANRIL in endothelial cells. Scale bars = 25 µm. Data were shown as mean ± SD (n = 3). (Student’s t test, *P < 0.05 versus control).

Fig 3: Mitochondrial morphological changes, apoptosis and related protein expression in HK-2 cells in hypoxia. A, In HK-2 cells treated with or without CoCl2, IF staining was performed with an anti-Drp1 antibody and MitoTracker Red. The yellow dots represent the colocalization of Drp1 and mitochondria. B, Quantitative analysis of mitochondrial fragmentation was performed. C, Confocal images of MitoSox Red and TMRE staining indicating mitochondrial ROS generation and the mitochondrial membrane potential (??m), respectively. D, Quantitative analyses of MitoSox Red and TMRE staining. E, Western blot analysis of HIF-1a, Mfn1, Mfn2, Drp1, p-Drp1(S-616) and Fis1 expression in HK-2 cells in normoxia or hypoxia. (F) Bar graph showing the quantitative analysis results of the relative band densities. G and H, Representative Western blot bands (G) and quantitative analysis (H) of mito-Cyt.C, mito-Bax, cyto-Cyt.C, cyto-Bax, c-caspase-9 and c-caspase-3. *P < .05 vs the normoxia group. n = 3

Fig 4: EI24 Promotes ER-mitochondria Contacts by VDAC2. a Western blot (WB) analysis of subcellular fractions from mouse liver. H homogenate; Mc crude mitochondrial fraction; Mp pure mitochondrial fraction; ER endoplasmic reticulum; MAM mitochondria-associated membrane; C cytosol. RTN4 Reticulon 4; ATL3 Atlastin 3; Mfn2 mitofusin-2. b U2OS cells that stably expressed CFP-ER and overexpressed the vector, 3× Flag-EI24 or 3× Flag-EI24?C were subjected to PLA (red) with anti-TOM20 and anti-GFP antibodies. DNA was stained with DAPI (blue). Scale bar, 10 µm. c Quantification of the number of PLA-positive puncta/cell in b. d Representative EM images of U2OS cells transfected with the vector, 3× Flag-EI24 or 3× Flag-EI24?C. Boxed areas at the top are magnified at the bottom, and the arrowheads indicate the ER-mitochondria contacts (<30 nm). ER endoplasmic reticulum; M mitochondrion. Scale bar, 500 nm. e Quantification of the ratio (%) of ER-mitochondria contacts (<30 nm) to the mitochondrial surface area (left) and the average number of close contacts per mitochondrion (right) in d (n = 118–311 mitochondria). f U2OS cells that stably expressed CFP-ER were co-transfected with 3× Flag-EI24 and control (shControl) or VDAC2 shRNA (shVDAC2) and subjected to PLA. Scale bar, 10 µm. g Quantification of the number of PLA-positive puncta/cell in f

Fig 5: Cordycepin stimulated mitochondrial fusion and enhanced Mfn2 expression in ischemic/reperfused hearts. (A) Representative transmission electron microscopic images of mitochondria in heart tissues and quantitative analysis of mitochondria length and the number of mitochondria per um2. (B) Quantitative RT-PCR analysis of Mfn2 mRNA expression level in mouse heart. (C) Western blotting and quantitative analysis of Mfn2 protein expression levels in heart tissues of mouse. DM, diabetes mellitus; MI/R, myocardial ischemia/reperfusion; Sham, sham-operated control rats; Cor, cordycepin. Scale bar, 1 µm *p < 0.05, **p < 0.01. n = 4 mice.