Fig 1: High glucose induced dysregulated cell function and aberrant expression of MFN1. (A) MFN1 in sections of lung tissues from adjacent normal, LAD and LAD+DM patients. Brown colour indicates positive staining. (B and C). Cell viability was evaluated by the CCK-8 assay in A549 and NCL-H292 cells. (D-E) The protein expression of MFN1 was determined by Western blot analysis in A549 cells (D) and in NCL-H292 cells (E). (F) A549 cells were stained with MFN1 antibody (red) and co-stained with TOM20 antibodies to visualize mitochondria (green) and DAPI staining was used to visualize cell nuclei (blue). Scale bar = 100 μm. Data shown are mean ± SEM, **P < 0.01 (n =6).Abbreviations: LAD, lung adenocarcinoma; DM, diabetes mellitus; NO, no glucose; LG, low glucose; NG, normal glucose; HG, high glucose. MFN1, mitofusin1; TOM20, mitochondrial import receptor subunit TOM20; DAPI, 4ʹ,6-diamidino-2-phenylindole.
Fig 2: ZFP36L2 knockdown suppresses hypoxia/reoxygenation (H/R) injury and alleviates mitochondrial fusion and fission in vitro.A A H/R injury model was established by inducing hypoxia for 4 h followed by reoxygenation for 1 h. The mRNA and protein expression of ZFP36L2 in cardiomyocytes were determined by qRT-PCR and western blot, respectively (n = 3). B Representative micrographs of cardiomyocytes after H/R injury expressing normal control shRNA or shRNA against ZFP36L2 stained with MitoTracker Red (mitochondria) and 4′, 6-diamidino-2-phenylindole (DAPI) (nucleus). Quantification of cells with fragmented mitochondria was detected under different conditions. At least 100 cells were counted per condition (Scale bar = 20 μm) (n = 3). C The levels of ROS, ATP and OCR were measured to evaluate mitochondrial function in H/R-treated cardiomyocytes, which was transfected with ZFP36L2 shRNA or shRNA NC. JC-1staining (2 µg/ml) was used to analyze mitochondrial membrane potential (△Ψm) (Scale bar = 50 μm) (n = 3). D Expression of mitochondrial fusion-related genes Drp1, Fis1, Mff, Mfn1, and Mfn2 after cardiomyocytes H/R treatment was evaluated by western blot (n = 3). E Representative micrographs from immunofluorescence assays of cardiomyocytes stained for Drp1 (green) and the nucleus (DAPI) (Scale bar = 100 μm) (n = 3). **P < 0.01; ***P < 0.001.
Fig 3: MFN1 was an important factor for EMT in human LAD. (A). The efficiency and specificity of siRNAs targeting MFN1 knockdown. The results showed that siMFN1 decreased the expression of MFN1 compared with that in cells treated with non-targeted control siRNA (NC). (B-C). The expression of mesenchymal and epithelial cell protein markers in A549 cells after high glucose treatment. (D and E). High glucose enhanced the expression of N-cadherin (red) and decreased the expression of E-cadherin (green) in the cytoplasm of A549 cells. Scale bars = 100 µm. Data shown are mean ± SEM from at least three separate experiments. *P < 0.05, **P < 0.01 vs NG+NC group. #P < 0.05, ##P < 0.01 vs HG+NC group significantly different as indicated.Abbreviations: EMT, epithelial-to-mesenchymal transition; LAD, lung adenocarcinoma; NC, non-targeted control; NG, normal glucose; HG, high glucose. MFN1, mitofusin1; siMFN1, small interfering RNA of MFN1; DAPI, 4ʹ,6-diamidino-2-phenylindole.
Fig 4: Glucose enhanced the interaction of MFN1 and Pink in A549 cells. (A) Predicted protein-protein interactions with MFN1 according to the STRING database. (B and C) The expression levels of Pink, Parkin and Fis1 were examined by Western blot in A549 cells after exposure to glucose for 24 hours. (D) Immunofluorescence of A549 cells from NG+NC, HG+NC, and HG+siMFN1. Cells were stained with DAPI (blue) and Pink (red). (E) A549 cells were exposed to 25 mM glucose, and whole cell lysates were extracted for co-immunoprecipitation with anti-Pink, and subsequent probing with anti-MFN1. Scale bar = 100 µm. Data shown are mean ± SEM. **P < 0.01 vs NG+NC group. #P < 0.05 vs HG+NC group (n = 4).Abbreviations: NC, non-targeted control; NG, normal glucose; HG, high glucose. MFN1, mitofusin1; siMFN1, small interfering RNA of MFN1; Pink, PTEN induced kinase; DAPI, 4ʹ,6-diamidino-2-phenylindole. IB, immunoblotting; IP, immunoprecipitation; STRING, https://string-db.org/.
Fig 5: MFN1 mediated the function of A549 cells mainly through Pink. (A and B) Western blot analysis was used to detect the expression of key EMT-related proteins in A549 cells after transfection with MFN1 overexpression plasmid and/or siPink. (C) The survival rate of A549 cells was studied by CCK-8 assay. (D and E) Transwell assays were used to evaluate cell infiltration capacity following overexpression of MFN1 and knockdown of Pink. (F) Representative images are presented to indicate the cellular localization patterns of the mRFP-GFP-LC3 fusion protein; scale bar = 50 μm. Data shown are mean ± SEM. **P < 0.01, ***P < 0.001 vs NG+NC group. #P < 0.05, ##P < 0.01 vs HG+NC group. $$P < 0.01, $$$P < 0.001 vs HG+MFN1 group (n = 4).Abbreviations: EMT, epithelial-to-mesenchymal transition; NC, non-targeted control; NG, normal glucose; HG, high glucose. MFN1, mitofusin1; si PINK, small interfering RNA of PTEN induced kinase; LC3B, microtubule-associated proteins 1A/1B light chain 3B; eGFP, enhanced green fluorescent protein; mRFP, monomer red fluorescent protein.
Supplier Page from Abcam for Anti-Mitofusin 1 antibody