Fig 1: Sustained hypermethylation of Mfn2 promoter DNA in retinal endothelial cells after removal of high glucose, and intervention with Aza. HRECs incubated in 20 mM glucose (HG) for four days, followed by 5 mM D-glucose (NG) for four days, in the presence or absence of 1 µM Aza were analyzed for (a) methylated cytosine at Mfn2 promoter DNA by Methylated DNA capture method and (b,c) Dnmt1 and Sp1 binding by ChIP technique. The values are plotted relative to those obtained from the ‘input DNA’ internal control. (d) Mfn2 mRNA was quantified by qPCR, and the values are represented as mRNA of Mfn2, relative to that of ß-actin, in each sample. Values from NG group values are adjusted to 1. (e) Mitochondrial localization of Mfn2 by immunofluorescence technique using Alexa-Flour 488 (green) and Texas Red (red) conjugated secondary antibodies for Mfn2 and CoxIV respectively. The images were captured in ZEISS 40X objective magnification with Apotome module. Pearson’s correlation coefficient was determined using co-localization software module. Data are represented as mean ± SD of the values obtained from four cell preparations, and each measurement done in duplicate. HG-NG and HG-NG/Aza = HRECs in 20 mM D-glucose for four days followed by 5 mM glucose, without or with Aza respectively, for four additional days; HG/Aza and NG/Aza= HRECs in continuous 20 mM or 5 mM glucose respectively, in the presence of Aza; L-Gl = 20 mM L-Glucose. *and #p < 0.05 compared to NG or HG respectively.
Fig 2: ERO1a knockdown alleviates mitochondrial dysfunction by reducing endoplasmic reticulum stress following H/R injury. (A) Cyt c expression levels in the cytoplasm and mitochondria were analyzed using western blotting. (B) ??m was detected using a microplate reader following staining with JC-1. Data are presented as the mean ± SD from three independent experiments. ***P<0.001 vs. CON group; ##P<0.01 vs. H/R group. ERO1a, endoplasmic reticulum oxidase 1a; CON, control; Cyt c, cytochrome c; H/R, hypoxia/reoxygenation; Cyto, cytoplasmic; Mito, mitochondria; COX IV, cyt c oxidase subunit IV; ??m, mitochondrial membrane potential; shRNA, short hairpin RNA; ns, not significant; TM, tunicamycin.
Fig 3: Both renal IPC and Tat-BECN1 preconditioning enhance mitophagy in proximal tubules during subsequent renal IRI in mice. (a) C57BL/6 mice were subjected to: (1) sham (n = 3); (2) I-R (n = 7); (3) IPC + I-R (n = 10). Kidneys were collected for immunoblot analysis of PINK1, COX4I1, IMMT/MIC60, and TOMM20. PPIB (peptidylprolyl isomerase B) was used as a loading control. Mito-QC mice were subjected to: (1) sham (n = 3); (2) IPC (n = 3); (3) I-R (n = 4); (4) IPC + I-R (n = 5). Kidneys were collected to determine mitophagy flux by fluorescence microscopy. (b) Representative images of the formation of mitolysosomes. Scale bar: 20 µm for low magnification and 5 µm for high magnification. (c) Quantitative analysis of the number of mitolysosomes per 400× field (renal cortex and outer stripe of outer medulla, glomeruli excluded). Data are expressed as mean ± SD. *, P < 0.05, significantly different from the sham group; #, P < 0.05, significantly different from I-R group. Furthermore, mito-QC mice were pretreated with Tat-BECN1 and its control peptide (Tat-Scramble) at a single dose of 20 mg/kg i.p. injection. Four h after preconditioning, mice were subjected to sham surgery or 27-min bilateral renal ischemia followed by 48 h of reperfusion (n = 3 for each). Kidneys were collected to determine mitophagy flux by fluorescence microscopy. (d) Representative images of the formation of mitolysosomes. Scale bar: 20 µm. (e) Quantitative analysis of the number of mitolysosomes per 400× field (renal cortex and outer stripe of outer medulla, glomeruli excluded). Data are expressed as mean ± SD. *, P < 0.05, significantly different from the sham group; #, P < 0.05, significantly different from Tat-Scramble + I-R group.
Fig 4: Activated BID is translocated to ischemic neurons following reperfusion. Representative immunofluorescent images of tBID obtained from the wild-type parietal cortex relevant to the infarct border zone. (A) Immunofluorescence signal of tBID was weak and diffusive in the non-ischemic cortex (left). A wild-type cortex staining control not incubated with the primary anti-BID antibody 6 h following reperfusion is presented on the right. (B) tBID immunofluorescence signal was increased at 3 h (left) and 6 h (right) following reperfusion and assumed a punctuated pattern. (C) tBID immunofluorescence signal appeared weaker at 3 h (left) and 6 h (right) following remote ischemic postconditioning. (A-C) Magnification, ×400; scale bar, 10 µm. Arrows indicate tBID signals. (D) Double staining of tBID and mitochondria COXIV in cortical neurons from control group, 6 h following reperfusion. The overlapping image indicated that tBID was localized in the mitochondria (arrows mark the double-stained neurons). (E) Double staining of tBID and NeuN in the same treated cortex as D. The overlapping image indicated that enhanced tBID signals were detected in neurons (as marked by the arrows). (D and E) Magnification, ×1,000; scale bar, 10 µm. BID, B-cell lymphoma 2 homology 3 interacting-domain death agonist; tBID, truncated BID; R-0, remote ischemic postconditioning 0 min group; COXIV, cytochrome c oxidase IV; NeuN, neuronal nuclear antigen.
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