Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: A, B (A) Equal amounts of protein extracted from WT and mutant MEFs stably expressing pLenti‐Opa1 or pLenti‐Pgs1 where indicated were separated by SDS–PAGE, immunoblotted with indicated antibodies and quantified by densitometry (B) relative to tubulin. Data represent mean ± SD of four independent experiments. C Equal amounts of protein extracted from WT MEFs treated with 20 μM CCCP (30 min) or 16 μM 4Br‐A23187 (18 h) were separated by SDS–PAGE, immunoblotted with indicated antibodies. Tubulin was used as loading control. D Representative confocal images of MEFs knocked in for mTurquoise2‐Dnm1l by Crispr/Cas9 genome editing in WT (WT Drp1KI ) and Opa1 Crispr ( Opa1 Crispr‐DrpKI ) MEFs treated with non‐targeting (NT) or Pgs1 siRNA for 72 h. DRP1 (mTurquoise2, purple), mitochondria (mitoYFP, green). Scale bar = 20 µm. E Bar graph representation of DRP1 localized to mitochondria (blue) vs cytosol (green). Data represent mean ± SD of five replicates (193–1,062 cells per cell line), One‐way ANOVA. Source data are available online for this figure.

Article Snippet: Mitochondria were stained overnight at 4°C with anti‐TOMM40 (diluted 1:1,000; ProteinTech #18409‐1‐AP) primary antibody and DRP1 with α‐DLP1 primary antibody (diluted 1:1,000, BD # 611112).

Techniques: Mutagenesis, Stable Transfection, Expressing, SDS Page, CRISPR

Journal: EMBO Molecular Medicine

Article Title: High‐throughput screening identifies suppressors of mitochondrial fragmentation in OPA1 fibroblasts

doi: 10.15252/emmm.202013579

Figure Lengend Snippet: Equal amounts of protein extracted from total (T), cytosolic flow‐through (C), and mitochondrial eluate (M) from MEFs of the indicated genotypes stably expressing MitoTag (pMXs‐3XHA‐EGFP‐OMP25) obtained following mitochondrial immunocapture were separated by SDS–PAGE, immunoblotted with indicated antibody, and quantified by densitometry. Data represent mean ± SD of three independent experiments, One‐way ANOVA. Representative confocal images of MEFs of the indicated genotypes showing subcellular DRP1 distribution. Mitochondria (TOMM40, green), DRP1 labeled with anti‐DRP1 antibody (red) and nuclei (NucBlue, blue). Scale bar = 10 μm. MiD49 / 51 / Mff KO MEFs lack all 3 DRP1 receptors (MiD49, MiD51, and MFF). Bar graph representation of DRP1 localized to mitochondria (green) vs cytosol (blue). Data represent mean ± SD of three independent experiments (884–3,116 cells per cell line), unpaired t ‐test; * P < 0.05. Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected fragmentation with 5 μM carbonyl cyanide m‐chlorophenyl hydrazine (CCCP) for the indicated time points. Images were captured every hour for 18 h. Scale bar = 10 μm. Supervised ML mitochondrial morphology quantification using WT MEFs treated with 5 μM CCCP for 18 h (fragmented), untreated (normal), or treated with 10 μM CHX for 9 h (hypertubular) training sets. Data represent mean ± SD of three independent experiments (131–426 cells per cell line), One‐way ANOVA; * P < 0.05, ** P < 0.01, **** P < 0.0001, ns; not significant. FRAP fusion assay in MEFs of the indicated genotype (see Movies , , ). Scale bar = 10 μm. Quantification of mitoYFP signal intensity measured at 200 ms intervals in the photobleached area (green box) for the indicated time (seconds), represented as relative fold recovery post‐bleach. Data represent mean ± SEM of two independent experiments ( n = 18–52 cells per genotype), One‐way ANOVA. Representative confocal images of live cell imaging of MEFs of the indicated genotypes subjected hyperfusion (SiMH) with 10 μM cycloheximide (CHX) for the indicated time points. Images were captured every hour for 9 h. Mitochondrial morphology quantification of using WT MEFs treated with 5 μM CCCP for 18 h (fragmented), untreated (normal), or treated with 10 μM CHX for 9 h (hypertubular) training sets. Data represent mean ± SD of four independent experiments, (155–745 cells per cell line), One‐way ANOVA. Source data are available online for this figure.

Article Snippet: Mitochondria were stained overnight at 4°C with anti‐TOMM40 (diluted 1:1,000; ProteinTech #18409‐1‐AP) primary antibody and DRP1 with α‐DLP1 primary antibody (diluted 1:1,000, BD # 611112).

Techniques: Stable Transfection, Expressing, SDS Page, Labeling, Live Cell Imaging, Single Vesicle Fusion Assay

Journal: Antioxidants

Article Title: Pimozide and Imipramine Blue Exploit Mitochondrial Vulnerabilities and Reactive Oxygen Species to Cooperatively Target High Risk Acute Myeloid Leukemia

doi: 10.3390/antiox10060956

Figure Lengend Snippet: IB suppresses phosphorylation of DRP1 in Flt3-ITD + cells. ( A ) A panel of genes related to the ER–mitochondria interface were assayed by real-time qRT-PCR. The panel included Fis1, Ip3r, Mfn1, Drp1, Grp75, Mtorc2, Vdac1, and Bap31 ( n = 3 for all except Drp1 where n = 7). p values were calculated relative to OCI-AML3 cells. ( B ) Western blot analysis for total Drp1 protein with β-actin as the loading control. ( C ) Western blot analysis for phospho-Drp1 protein with β-actin as the loading control. ( D , E ) Densitometry analysis was performed and quantitation is shown for replicates of the Western blots in panels C and D. n = 3 and p values were calculated with comparisons as shown between either OCI-AML3 cells or with or without treatment. * p < 0.05; ** p < 0.01.

Article Snippet: Antibodies for Western blot were obtained from the following: Anti-STAT5 (Phospho-STAT5 Y694) antibody (Abcam, Cambridge, UK), Phospho-Akt (Ser473) (D9E), Phospho-Akt (Thr308) (D25E6) (Cell Signaling), Phospho-DRP1 (Ser637) (D3A4) (Cell Signaling, Danvers, MA, USA), DRP1 Antibody (Novus Biologicals), p38alpha MAPK antibody and Phospho-p38alpha MAPK (Cell Signaling, Danvers, MA, USA).

Techniques: Phospho-proteomics, Quantitative RT-PCR, Western Blot, Control, Quantitation Assay

Journal: PLoS ONE

Article Title: Novel Roles of Epoxyeicosanoids in Regulating Cardiac Mitochondria

doi: 10.1371/journal.pone.0160380

Figure Lengend Snippet: A. 3D images of mitochondria were analyzed morphometrically to identify mitochondrial structures and to numerically report their length and size. Mitochondria were stained with MitoTracker Red (100nM) and Z-stacks images were captured with Zeiss AxioObserver Epifluorescence microscope. Image processing was conducted by Zeiss ZEN 12 and Bitplane Imaris software as detailed in the methods section. B. Distribution of mitochondria according to their length into long (>5μm), short (<5μm) and fragmented (<0.5μM) mitochondria. To verify the morphometric method in our cells, DRP1-dependent fission was inhibited by forskolin (20μM) and cyclosporine A (10μM) or activated by H89 (1μM) and iononmycin (5μM). C. The ratio of long mitochondria to short and fragmented mitochondria provides a single descriptor to mitochondrial length allowing simple statistical analysis between treatment groups. Values represent mean+SEM; N = 3 independent experiments, *, p< 0.05 in comparison to control.

Article Snippet: Membranes were washed three times with TBS-T buffer (with 15-min intervals) and then incubated with either mouse monoclonal antibody against cytochrome c oxidase subunit 4 (Cell Signaling Tech., Inc., Whitby, ON, cat#11967), rabbit polyclonal antibody against succinate dehydrogenase subunit A (Cell Signaling Tech., Inc., Whitby, ON, cat#5839), mouse monoclonal antibody against prohibitin (Fitzgerald #10R-P140A), rabbit monoclonal antibody against DRP1 (Cell Signaling Tech., Inc., Whitby, ON, cat#8570), rabbit polyclonal antibody against phospho-DRP1 (Ser637) (Cell Signaling Tech., Inc., Whitby, ON, cat#4867), rabbit polyclonal antibody against Fis1 (Novus Biologicals Canada, ULC, Oakville, ON, cat#IMG-5113A), rabbit monoclonal antibody against Mfn2 (Cell Signaling Tech., Inc., Whitby, ON, cat#9482), mouse monoclonal antibody against OPA1 (BD Biosciences, Mississauga, ON, cat#612606) or rabbit polyclonal antibody against α-tubulin (Abcam., Cambridge, UK, cat#4074) overnight at 4°C.

Techniques: Staining, Microscopy, Software, Comparison, Control

Journal: PLoS ONE

Article Title: Novel Roles of Epoxyeicosanoids in Regulating Cardiac Mitochondria

doi: 10.1371/journal.pone.0160380

Figure Lengend Snippet: HL-1 cardiac cells were treated under normal cell culture conditions (NS Ctrl) or following 24h starvation (Starv) with or without UA-8 (1μM) and/or 14,15-EEZE (10μM). ( A ) Representative images of mitochondrial morphology demonstrating starvation induced mitochondrial hyperfusion that was inhibited by UA-8. ( B ) The ratio of long:short mitochondria reflecting the starvation-induced hyperfusion and the inhibitory effect of UA-8 (>25 cells per treatment group were assessed in each experiment). ( C ) Relative expression of the mitochondrial fission protein DRP1. ( D ) Relative expression of the mitochondrial fission protein Fis1. ( E ) Relative expression of the mitochondrial fusion protein Mfn2. Values represent mean+SEM; N = 3 independent experiments, *, p< 0.05 in comparison to non-starvation control, and, #, p< 0.05 between indicated groups.

Article Snippet: Membranes were washed three times with TBS-T buffer (with 15-min intervals) and then incubated with either mouse monoclonal antibody against cytochrome c oxidase subunit 4 (Cell Signaling Tech., Inc., Whitby, ON, cat#11967), rabbit polyclonal antibody against succinate dehydrogenase subunit A (Cell Signaling Tech., Inc., Whitby, ON, cat#5839), mouse monoclonal antibody against prohibitin (Fitzgerald #10R-P140A), rabbit monoclonal antibody against DRP1 (Cell Signaling Tech., Inc., Whitby, ON, cat#8570), rabbit polyclonal antibody against phospho-DRP1 (Ser637) (Cell Signaling Tech., Inc., Whitby, ON, cat#4867), rabbit polyclonal antibody against Fis1 (Novus Biologicals Canada, ULC, Oakville, ON, cat#IMG-5113A), rabbit monoclonal antibody against Mfn2 (Cell Signaling Tech., Inc., Whitby, ON, cat#9482), mouse monoclonal antibody against OPA1 (BD Biosciences, Mississauga, ON, cat#612606) or rabbit polyclonal antibody against α-tubulin (Abcam., Cambridge, UK, cat#4074) overnight at 4°C.

Techniques: Cell Culture, Expressing, Comparison, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Intermittent Fasting Alleviates Anesthesia/Surgery‐Induced Delirium‐Like Behavior in Aged Mice by Remodeling Gut Microbiota

doi: 10.1002/cns.70748

Figure Lengend Snippet: Intermittent fasting protects against anesthesia/surgery‐induced mitochondrial fission and the reduction of ATP production in the hippocampus. (a) Transmission electron microscopy images of mitochondrial morphology in neurons of mice in ad libitum (AL) and intermittent fasting (IF) groups, exposed to anesthesia/surgery (AS) or control (C) conditions. Scale bar = 2.0 μm. Red arrows indicate mitochondria. (b) Representative western blot images of β‐Actin and VDAC expression in total protein, cytosolic fraction, and mitochondrial fraction of mice. (c) Representative western blot images of DRP1 levels in hippocampal mitochondrial fractions of mice ( n = 4 mice/group). (d) Representative western blot images of AMPK/PGC1α signaling pathway in the hippocampus of mice. (e) Quantitative analysis of DRP1 levels in hippocampal mitochondrial fractions and AMPK/PGC1α signaling in hippocampal tissues from immunoblots ( n = 4 mice/group). (f) ATP levels measured in hippocampal tissues of mice ( n = 5 mice/group). (g) Mitochondrial membrane potential levels in hippocampal tissues of mice ( n = 5 mice/group). Results are presented as mean ± standard error of the mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: We used primary antibodies against DRP1 (1:1000; 8570 T, Cell Signaling, Danvers, MA, USA), AMPKα (1:1000; 25,325, Cell Signaling), and p‐Thr172‐AMPK (1:1000; 2531S, Cell Signaling) and corresponding secondary antibodies conjugated to horseradish peroxidase (1:10,000; 511,203, ZenBio, Chengdu, China).

Techniques: Transmission Assay, Electron Microscopy, Control, Western Blot, Expressing, Membrane

Journal: CNS Neuroscience & Therapeutics

Article Title: Intermittent Fasting Alleviates Anesthesia/Surgery‐Induced Delirium‐Like Behavior in Aged Mice by Remodeling Gut Microbiota

doi: 10.1002/cns.70748

Figure Lengend Snippet: Gut microbiota influence hippocampal synaptic ultrastructure and mitochondrial dynamics post‐transplantation. (a) Representative transmission electron microscopy images of synaptic ultrastructure of mice that received fecal microbiota transplant (F) from ad libitum (AL) or intermittent fasting (IF) group mice exposed to anesthesia/surgery (AS). (b) Measurements of postsynaptic density (PSD) length and width ( n = 5 slices/group). Scale bar = 1.0 μm (c, d) Western blot images and quantification of PSD95 levels in the hippocampus of mice ( n = 4 mice/group). (e) Transmission electron microscopy images of mitochondrial morphology of mice. Scale bar = 2.0 μm. Red arrows indicate mitochondria. (f) Quantitative analysis of DRP1 levels in hippocampal mitochondrial fractions and AMPK/PGC1α signaling in hippocampal tissues of mice from immunoblots ( n = 4 mice/group). (g) Representative western blot images of AMPK/PGC1α signaling pathway in the hippocampus of mice. (h) Representative western blot images of DRP1 levels in hippocampal mitochondrial fractions of mice ( n = 4 mice/group). (i) ATP levels measured in hippocampal tissues of mice ( n = 5 mice/group). (j) Mitochondrial membrane potential levels in hippocampal tissues of mice ( n = 5 mice/group). Results are presented as mean ± standard error of the mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: We used primary antibodies against DRP1 (1:1000; 8570 T, Cell Signaling, Danvers, MA, USA), AMPKα (1:1000; 25,325, Cell Signaling), and p‐Thr172‐AMPK (1:1000; 2531S, Cell Signaling) and corresponding secondary antibodies conjugated to horseradish peroxidase (1:10,000; 511,203, ZenBio, Chengdu, China).

Techniques: Transplantation Assay, Transmission Assay, Electron Microscopy, Western Blot, Membrane

Journal: CNS Neuroscience & Therapeutics

Article Title: Intermittent Fasting Alleviates Anesthesia/Surgery‐Induced Delirium‐Like Behavior in Aged Mice by Remodeling Gut Microbiota

doi: 10.1002/cns.70748

Figure Lengend Snippet: Short‐chain fatty acid supplementation protects hippocampal synaptic integrity and mitochondrial function post‐anesthesia/surgery. (a) Representative transmission electron microscopy images of synaptic ultrastructure of mice that SCFA‐supplemented mice (SCFAs+AS) compared to controls (Con+AS) post‐surgery. Scale bar = 1.0 μm (b) Measurements of postsynaptic density (PSD) length and width ( n = 5 slices/group). (c, d) Western blot images and quantification of PSD95 levels in the hippocampus of mice ( n = 4 mice/group). (e) Transmission electron microscopy images of mitochondrial morphology of mice. Scale bar = 2.0 μm. Red arrows indicate mitochondria. (f, g) Western blot images and quantification of DRP1 levels in hippocampal mitochondrial fractions ( n = 4 mice/group). (h) ATP levels measured in hippocampal tissues of mice ( n = 5 mice/group). (i) Mitochondrial membrane potential levels in hippocampal tissues of mice ( n = 5 mice/group). Results are presented as mean ± standard error of the mean (SEM). * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: We used primary antibodies against DRP1 (1:1000; 8570 T, Cell Signaling, Danvers, MA, USA), AMPKα (1:1000; 25,325, Cell Signaling), and p‐Thr172‐AMPK (1:1000; 2531S, Cell Signaling) and corresponding secondary antibodies conjugated to horseradish peroxidase (1:10,000; 511,203, ZenBio, Chengdu, China).

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Membrane

Journal: International journal of molecular sciences

Article Title: Novel Relationship between Mitofusin 2-Mediated Mitochondrial Hyperfusion, Metabolic Remodeling, and Glycolysis in Pulmonary Arterial Endothelial Cells.

doi: 10.3390/ijms242417533

Figure Lengend Snippet: Figure 1. Effect of Mfn2 overexpression on fusion and fission mediators, apoptosis, and proliferation in pulmonary arterial endothelial cells. PAECs were transduced with Ad-MFN2 (MOI = 5) or Ad- GFP, and Western blot analysis was used to confirm increased Mfn2 protein levels ((A), upper blot). The membrane was stripped and reprobed with an Mfn1 antibody to analyze the expression of Mfn1 protein. Overexpression of Mfn2 increased Mfn1 protein levels ((A), middle blot). Mfn2 overexpression did not affect the expression level of the fusion mediator Opa1 (B) or the fission mediators Drp1 (C) and Fis1 (D). Representative images are shown. β-actin was used to normalize protein loading. Mfn2 overexpression increased the aspect ratio (AR) in PAECs (E). TUNEL staining of PAECs identified increased apoptosis in MFN2-overexpressing PAECs (F). MFN2 overexpression also reduced the proliferation of PAECs (G). Data are mean ± SEM; n = 4–6. * p < 0.05 vs. control PAECs.

Article Snippet: For Western blots, a primary monoclonal Mfn2 antibody (#11925S, Cell Signaling Technologies, Danvers, MA, USA), a primary polyclonal Mfn1 antibody (#PA5-79665, Thermo Fisher Scientific, Waltham, MA, USA), a primary monoclonal Opa1 antibody (#612606, BD Biosciences, Franklin Lakes, NJ, USA), a primary monoclonal Drp1 antibody (#5391S, Cell Signaling Technologies, Danvers, MA, USA), a primary monoclonal Fis1 antibody (#32525S, Cell Signaling Technologies, Danvers, MA, USA), a primary monoclonal HIF-1α antibody Int.

Techniques: Over Expression, Transduction, Western Blot, Membrane, Expressing, TUNEL Assay, Staining, Control