Journal: Advanced Science

Article Title: Cytoplasmic Escape of Mitochondrial DNA Mediated by Mfn2 Downregulation Promotes Microglial Activation via cGas‐Sting Axis in Spinal Cord Injury

doi: 10.1002/advs.202305442

Figure Lengend Snippet: Mitochondria swelling and expression of mitofusin decreased after SCI. a) Representative ultrastructure of microglia in the perilesional tissues at 7 d after SCI (Scale bar: 1 µm (a1,a2) and 0.5 µm (a3,a4)). b) Western blot analysis of the Drp1, Mfn1, and Mfn2 levels in microglia of Control and LPS. c) Western blot analysis of cGas, Sting, Drp1, and Mfn2 levels in microglia of Control and LPS, LPS+MASM7, and LPS+Mdivi‐1. d) Representative of immunofluorescence staining of M1 state (iNOS) in microglia (Scale bar: 20 µm). e) Levels of pro‐inflammatory cytokines, including IL‐1β, IFN‐β, and TNF‐α in the medium of microglia. f) Flow cytometric analysis on the expression levels of M1 ratio (F4/80/ CD86+). g) The neuron death rate measured by Annexin V and PI staining. n = 6 for each group. h) Mfn2 /Iba1 double immunostaining in the perilesional tissues 7 d after SCI (Scale bar: 50 µm). Error bars denote mean ± SEM, ns, no significance, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: [ ] To activate Mfn2, microglia were treated with 1 μ m MASM7 (MedChemExpress, USA).

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Staining, Double Immunostaining

Journal: Advanced Science

Article Title: Cytoplasmic Escape of Mitochondrial DNA Mediated by Mfn2 Downregulation Promotes Microglial Activation via cGas‐Sting Axis in Spinal Cord Injury

doi: 10.1002/advs.202305442

Figure Lengend Snippet: Mitochondrial fission‐induced mtDNA release mediates activation of Sting signaling in LPS‐induced microglial. a) Representative of immunofluorescence staining of Mfn2 in microglia in different groups (Scale bar = 20 µm). b) Western blot and quantitative analysis of Mfn2, cGas, Sting, Irf3, p‐Irf3, P65 and p‐P65 in microglia of Control, LPS, LPS + vehicle, and LPS + MASM7. c) dsDNA and HSP60 double immunostaining microglia in different groups (Scale bar: 20 µm). d) Representative MitoTracker fluorescence images illustrating mitochondrial morphology in microglia (Scale bar: 20 µm). e) Representative fluorescence staining of JC‐1 aggregates (red)/JC‐1 monomers (green) illustrating the MMP (Scale bar: 20 µm). f) Representative MitoSOX fluorescence images of mitochondria‐derived ROS (Scale bar: 20 µm). Error bars denote mean ± SEM, ns, no significance, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: [ ] To activate Mfn2, microglia were treated with 1 μ m MASM7 (MedChemExpress, USA).

Techniques: Activation Assay, Immunofluorescence, Staining, Western Blot, Control, Double Immunostaining, Fluorescence, Derivative Assay

Journal: Advanced Science

Article Title: Cytoplasmic Escape of Mitochondrial DNA Mediated by Mfn2 Downregulation Promotes Microglial Activation via cGas‐Sting Axis in Spinal Cord Injury

doi: 10.1002/advs.202305442

Figure Lengend Snippet: Schematic process of the activation of cGas‐Sting signaling mediated by Mfn2‐dependent release of mtDNA in microglia in SCI.

Article Snippet: [ ] To activate Mfn2, microglia were treated with 1 μ m MASM7 (MedChemExpress, USA).

Techniques: Activation Assay

Journal: Antioxidants (Basel, Switzerland)

Article Title: The Effect of Astaxanthin on Mitochondrial Dynamics in Rat Heart Mitochondria under ISO-Induced Injury.

doi: 10.3390/antiox12061247

Figure Lengend Snippet: Figure 4. The influence of AX and ISO change of DRP1, Mfn2, and OPA1 in RHM. (a) upper part—Western blot with antibody to DRP1. Tom20 was used for normalization of proteins. Lower part—quantitative characteristic presented as the ratio of DRP1 to Tom20; (b) upper part—Western blot with antibody to Mfn2, lower part—quantitative characteristic presented as the ratio of Mfn2 to Tom20; (c) upper part—Western blot with antibody to OPA1, lower part—quantitative characteristic presented as the ratio of OPA1 to Tom20. The data are presented as the means ± SDs of four independent experiments. * p < 0.05 significant values compared with control (group 1); # p < 0.05 significant values relative ISO to (group 3).

Article Snippet: The membranes were blocked in Roti-block solution (Carl Roth GmbH + Co., Karlsruhe, Germany) for 1 h. The membranes were stained with the polyclonal antibodies to DRP1 (Elabscience, Houston, TX, USA) at dilution 1:2000, Mfn2 (Elabscience, Houston, TX, USA) at dilution 1:2000, OPA1 (Cloud-Clone Corp., Katy, TX, USA) at dilution 1:1000, PHB2 (Cusabio, Houston, TX, USA) at dilution 1:1000, and PINK1 (Cusabio, Houston, TX, USA) dilution 1:500 overnight.

Techniques: Western Blot, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Increased ROS-Dependent Fission of Mitochondria Causes Abnormal Morphology of the Cell Powerhouses in a Murine Model of Amyotrophic Lateral Sclerosis

doi: 10.1155/2021/6924251

Figure Lengend Snippet: Primary and secondary antibodies used for Western blotting.

Article Snippet: Anti-MFN2 mouse polyclonal IgG antibody , 1 : 100 in Roti-TBS (#1060.1, Roth, Germany) , #ARP89255_P050, Aviva systems biology, USA.

Techniques: Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Increased ROS-Dependent Fission of Mitochondria Causes Abnormal Morphology of the Cell Powerhouses in a Murine Model of Amyotrophic Lateral Sclerosis

doi: 10.1155/2021/6924251

Figure Lengend Snippet: Abnormal fission-related proteins in cervical wobbler spinal cord at p40. (a) mRNA expression levels of Mfn1 , Mfn2 , Opa1 , Oma1 , and Dnm1l from the stable phase of wild-type (WT) and wobbler (WR) spinal cords were investigated by qPCR. mRNA levels were significantly reduced in WR except Dnm1l . For relative quantification, the 2 −∆∆Ct method was conducted using GAPDH for normalization. N = 7-11 per genotype. (b) Exemplary Western blots of Mfn1 (≈85 kDa), Mfn2 (≈85 kDa), Opa1 (≈100 kDa), and Oma1 (≈50 kDa) in the cervical spinal cord of p40 WT and WR. Actin (≈45 kDa) and calnexin (≈90 kDa) were used as control proteins. Bar charts represent the semiquantitative analysis of protein expression levels. Western blots revealed unchanged expression of Mfn1, Opa1, and Oma1 as well as significantly increased expression of Mfn2 in the cervical spinal cord of wobbler mice. N = 8-10 per genotype. (c) Exemplary Western blots of Drp1 (≈85 kDa), p-Drp1 (Ser616; ≈85 kDa), and actin (≈45 kDa) as control protein. Analysis of band intensity is presented in bar charts. Total amount of Drp1 does not differ between the two genotypes; Drp1 is significant more often phosphorylated at Ser616 in cervical spinal cords of wobbler mice. N = 9 per genotype. (d) Exemplary Western blots of CaMKII (≈55 kDa) and oxidized Ox-CaMKII (Met281/282; ≈55 kDa) in combination with calnexin (≈90 kDa) as control protein from wild-type and wobbler cervical spinal cords. Analysis of band intensity is presented in bar charts and showed equal CaMKII levels; thus, the proportion of oxidized CaMKII at Met281/282 is significantly increased in wobbler spinal cords compared to wild-type. N = 7 per genotype. All data are presented as the mean values ± SEM, and Student's t -test was performed for significance testing between WT and WR. Values with p < 0.05 were considered to be significant. Significant differences are indicated by ns > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Anti-MFN2 mouse polyclonal IgG antibody , 1 : 100 in Roti-TBS (#1060.1, Roth, Germany) , #ARP89255_P050, Aviva systems biology, USA.

Techniques: Expressing, Quantitative Proteomics, Western Blot, Control

Journal: Protein & Cell

Article Title: PKM2 coordinates glycolysis with mitochondrial fusion and oxidative phosphorylation

doi: 10.1007/s13238-019-0618-z

Figure Lengend Snippet: PKM2 binds to MFN2 . (A) Left panel: Silver-stained gel showing the proteins that interact with Strep-PKM2 in a pull-down assay in HepG2 cells. Right panel: The peptides from MFN2 that were identified by mass spectrometry analysis. (B) Co-immunoprecipitation (co-IP) showing the interaction of overexpressed PKM2 and MFN2 in HEK293T cells. (C) Co-IP showing the interaction of endogenous PKM2 and MFN2 in H1299 and HepG2 cells. (D) Deletion analysis of the regions in PKM2 is required for the PKM2:MFN2 interaction. Interaction-competent PKM2 polypeptides are indicated by (+) in each schematic. (E) Deletion analysis of the regions in MFN2 is required for the PKM2:MFN2 interaction. Interaction-competent MFN2 polypeptides are indicated by (+) in each schematic

Article Snippet: The phospho-S200 MFN2 antibody was generated and purified by AbMax Biotechnology.

Techniques: Staining, Pull Down Assay, Mass Spectrometry, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Protein & Cell

Article Title: PKM2 coordinates glycolysis with mitochondrial fusion and oxidative phosphorylation

doi: 10.1007/s13238-019-0618-z

Figure Lengend Snippet: mTOR phosphorylates MFN2 and enhances its binding to PKM2 . (A) Co-IP showing the interaction of endogenous PKM2 and MFN2 in H1299 cells in response to treatment with amino acids and/or Torin1. H1299 cells incubated with amino acid-free RPMI1640 for 6 h were exposed to 250 nmol/L Torin1 or control vehicle for another 2 h, and then treated with amino acids for 30 min. (B) Amino acid sequence alignment of MFN2 orthologs. The conserved serine residue (S, red) is phosphorylated by mTOR. (C) In vitro kinase assay showing phosphorylation of the His-tagged G/M fragment of MFN2 (which contains amino acids 97–610 of MFN2) and S6K by truncated mTOR. (D) Immunoblots of co-IP assays showing the relative association of PKM2 with WT MFN2 or MFN2 with a Ser-to-Ala or Ser-to-Glu mutation at position 200 (S200A or S200E) in transfected HEK293T cells in the presence or absence of amino acids

Article Snippet: The phospho-S200 MFN2 antibody was generated and purified by AbMax Biotechnology.

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Incubation, Control, Sequencing, Residue, In Vitro, Kinase Assay, Phospho-proteomics, Western Blot, Mutagenesis, Transfection

Journal: Protein & Cell

Article Title: PKM2 coordinates glycolysis with mitochondrial fusion and oxidative phosphorylation

doi: 10.1007/s13238-019-0618-z

Figure Lengend Snippet: Phosphorylation of MFN2 enhances mitochondrial OXPHOS and attenuates glycolysis . (A) Effect of PKM2 on the GTPase activity of WT MFN2 or mutants of MFN2. (B) Representative images and statistical analyses showing the effect of MFN2 and its mutants on mitochondrial morphology in H1299 cells in the presence or absence of Torin1 (250 nmol/L) for 4 h. (C–I) Effect of MFN2 and its mutants on OCR (C), relative PKM2 activity (D), ECAR (E), relative glucose consumption (F), relative lactate production (G), ratio of M+3 2-phosphoglycerate/3-phosphoglycerate (H) and M+2 citrate (I). Data are shown as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 6

Article Snippet: The phospho-S200 MFN2 antibody was generated and purified by AbMax Biotechnology.

Techniques: Phospho-proteomics, Activity Assay

Journal: Protein & Cell

Article Title: PKM2 coordinates glycolysis with mitochondrial fusion and oxidative phosphorylation

doi: 10.1007/s13238-019-0618-z

Figure Lengend Snippet: The phosphorylation status of MFN2 affects cancer cell growth . (A) Effect of MFN2 and its mutants on cell proliferation in H1299 cells. n = 6. (B and C) Representative images and statistical analyses showing the effect of MFN2 and its mutants on tumor size (B) or tumor weight (C) in nude mice. n = 10 mice. (D–G) Effect of MFN2 and its mutants on relative PKM2 activity (D), relative pyruvate level (E), relative lactate production (F) and relative G6P/F6P (glucose-6-phosphate/fructose-6-phosphate) level (G) in tumors from the xenograft assay. (H) Representative images and statistical analyses showing the effect of MFN2 and its mutants on mitochondrial morphology in tumors from the xenograft assay. Scale bars, 10 μm. (I) mTOR phosphorylates MFN2 at Ser200, which enhances the interaction of MFN2 with PKM2, promotes OXPHOS by increasing mitochondrial fusion, and attenuates glycolysis. Data are shown as mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The phospho-S200 MFN2 antibody was generated and purified by AbMax Biotechnology.

Techniques: Phospho-proteomics, Activity Assay, Xenograft Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: Inhibiting MARCH5/Mfn2 signaling as an alternative strategy to protect cardiomyocytes from hypoxia-induced mitochondrial dysfunction

doi: 10.1016/j.csbj.2025.07.001

Figure Lengend Snippet: Characterization and identification of MARCH5/Mfn2 PPIs ( A ) Structural overlay of the top five ranked models predicted by AlphaFold 3 for the complex between the Mfn2 cytosolic domain construct (residues 1–540, 654–757 of UniProt O95140 ) and full-length MARCH5 (UniProt Q9NX47 ). Mfn2 is shown as a gray cartoon. The five MARCH5 predictions are shown as cartoons colored by model rank (Model 0: red, Model 1: orange, Model 2: yellow, Model 3: green, Model 4: blue). The approximate position of the cytoplasmic face of the mitochondrial outer membrane (MOM) relative to Mfn2 is indicated by the black line. ( B ) Tables listing key Mfn2 and MARCH5 residues predicted by AlphaFold 3 to interact most frequently with full-length MARCH5 and Mfn2, respectivley, along with their ConSurf scores. These residues exhibit average to variable conservation scores (4−6) ( C ) The full structure of MARCH5 is schematically represented with an N-terminal Ring domain (RING, orange) and four transmembrane (TM, yellow) domains. ( D ) The linear structure of full Mfn2 includes the N-terminal GTPase domain, the heptad repeat 1 (HR1) domain, the proline-rich (PR) domain, the two TM domains, and the C-terminal HR2 domain, with the numbers indicating the amino acid residues of each domain. ( E ) Mfn2 binding curve to MARCH5 and control protein was plotted. Mfn2 was immobilized, and MARCH5 or the control protein, bovine serum albumin (BSA), was analyzed at increasing concentrations. The K D value of Mfn2 binding with BSA is undetectable (unstable). The Y-axis represents the response in biosensor units (BU), and the X-axis corresponds to the different concentrations of the analyte protein. Data are presented as mean ± SD (n = 3). ( F ) MARCH5 recombinant protein interacts with and ubiquitinates Mfn2 recombinant protein in vitro. Immunoprecipitation (IP) of MARCH5 was performed using Mfn2, and ubiquitination of Mfn2 was assessed through immunoblots with an anti-HA antibody (71–5500, Invitrogen/Thermo Fisher, Waltham, MA, USA). The Mfn2 and MARCH5 proteins were detected using anti-Mfn2 (ab56889, Abcam, UK) and anti-MARCH5 (ab185054, Abcam, UK) antibodies.

Article Snippet: Finally, Mfn2 and MARCH5 proteins were detected using anti-Mfn2 (ab56889, Zotal, Abcam) and anti-MARCH5 (ab185054, Zotal, Abcam) antibodies.

Techniques: Construct, Membrane, Binding Assay, Control, Recombinant, In Vitro, Immunoprecipitation, Ubiquitin Proteomics, Western Blot