Journal: Science translational medicine

Article Title: Ablation of the stress protease OMA1 protects against heart failure in mice.

doi: 10.1126/scitranslmed.aan4935

Figure Lengend Snippet: Fig. 4. Analysis of ISO response in vitro in neonatal cardiomyocytes. (A) Mitochondrial respirometry analysis of neonatal cardiomyocytes upon different treatments measured by Seahorse and maximum respiration rate (MRR) quantification. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FBS, fetal bovine serum; NAC, N-acetyl-l- cysteine; OCR, oxygen consumption rate. (B) 2′,7′-Dichlorofluorescein diacetate (H2-DCFDA)–mediated ROS quanti- fication in neonatal cardiomyocytes upon different treatments. (C) Fura-2-acetoxymethyl ester (Fura-2 AM)–mediated intramitochondrial calcium quantification in neonatal cardiomyocytes upon different treatments. (D) Tetramethyl- rhodamine methyl ester perchlorate (TMRM)–mediated mitochondrial membrane potential determination in neonatal cardiomyocytes upon different treatments. (E) Analysis of OPA1 processing by Western blot. (F) Analysis of MCU ex- pression by Western blot of mitochondrial lysate. SDHA and core2 are used as loading controls. (G) Kurtosis analysis as indicator of calcium distribution in the cardiomyocytes at the indicated treatments. Basal and final treatments corre- spond to time 0 and time 9 min, respectively. (H) Time-lapse images (40×) at the indicated time showing calcium (Fura-2 AM) and mitochondrial (MitoTracker Deep Red) distribution under different treatments. Scale bars, 20 M. Data in upper and middle panels of (A) are given as means ± SEM and in the rest of graphics as scatter dot plots, and lines are means ± SD. Differences assessed by two-way ANOVA and Sidak’s multiple comparison test (A) or one-way ANOVA and Tukey’s multiple comparison test (B to F). N as described in table S1; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The following antibodies were used: SDHA (Novex); ATP5B, OPA1, glyceraldehyde-3-phosphate dehydrogenase, core1, core2, and MnSOD (Abcam); MCU and M1CU1 (Sigma-Aldrich); and Tom20 (Santa Cruz Biotechnology).

Techniques: In Vitro, Membrane, Western Blot, Comparison

Journal: Science translational medicine

Article Title: Ablation of the stress protease OMA1 protects against heart failure in mice.

doi: 10.1126/scitranslmed.aan4935

Figure Lengend Snippet: Fig. 7. Heart-specific OMA1 down-regulation protects from hypertrophy induced by TAC. OPA1 processing analysis by Western blot (A) and quantification (B) in isolated mitochondria from heart and liver of mice injected with sh scrambled (shScr) or shOMA1 viral particles. Mitochondria were incubated in the presence of carbonyl cyanide m-chlorophenyl hydrazone for the indicated time points. MW, molecular weight; nt, not treated. (C) Des Ao Vel, heart weight/body weight (HW/BW), % EF, and rate of ATP synthesis in WT inbred (C57BL/6JOlaHsd) and outbred (CD1) mice injected with sh shScr or shOMA1 virus and then subjected to TAC. Analyses were performed 3 weeks after TAC surgery. (D) OPA1 processing analysis by Western blot (left) and quantification OPA1 (right) in mitochondrial heart fractions form WT in- bred (C57BL/6JOlaHsd) and outbred (CD1) mice injected with sh scr or sh OMA1 for the indicated conditions. Data are given as scatter dot plots, and lines are means ± SD. Differences assessed by one-way ANOVA and Tukey’s multiple comparison test. N as described in table S1; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The following antibodies were used: SDHA (Novex); ATP5B, OPA1, glyceraldehyde-3-phosphate dehydrogenase, core1, core2, and MnSOD (Abcam); MCU and M1CU1 (Sigma-Aldrich); and Tom20 (Santa Cruz Biotechnology).

Techniques: Western Blot, Isolation, Injection, Incubation, Molecular Weight, Virus, Comparison

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Topical Application of the Antimicrobial Agent Triclosan Induces NLRP3 Inflammasome Activation and Mitochondrial Dysfunction

doi: 10.1093/toxsci/kfaa056

Figure Lengend Snippet: Primary and Secondary Antibodies Used for ProteinSimple Wes Immunoassay

Article Snippet: Total protein was used as a loading control and for normalization. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Reference Sample Concentration Antibody Dilution Secondary Antibody Opa1 Novus Biologicals 0.2 mg/ml (skin) 1:200 (skin) Anti-rabbit-HRP (Wes detection kit) NB-110-55290SS 0.2 mg/ml (dLN) 1:200 (dLN) Drp1 Novus Biologicals 0.1 mg/ml (skin) 1:150 (skin) NB110-55288SS 0.1 mg/ml (dLN) 1:150 (dLN) Open in a separate window Primary and Secondary Antibodies Used for ProteinSimple Wes Immunoassay

Techniques: Concentration Assay

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Topical Application of the Antimicrobial Agent Triclosan Induces NLRP3 Inflammasome Activation and Mitochondrial Dysfunction

doi: 10.1093/toxsci/kfaa056

Figure Lengend Snippet: A–O, Triclosan induces mitochondrial morphology changes in draining lymph node (dLN) cells. Mitochondrial morphology in dLN cells was examined by transmission electron microscopy after dermal exposure to 0% (A–C), 0.75% (D, H), 1.5% (E, I), or 3% (F, G, J, K) triclosan for 4days. Representative images of triclosan-exposed mitochondria show signs of damage (D–G) as indicated by fragmented mitochondria and inhomogeneous electron transparent matrix (asterisk). Triclosan also induced a change in morphology with U-, C-, or ring-shaped mitochondria (H–K) (arrow head). Scale bars = 250nm. Morphometric analyses (n = at least 45 mitochondria) were done via Fiji ImageJ measuring mitochondrial circularity (L), roundness (M), and aspect ratio (N). Percent mitochondria that show standard mitochondrial morphology and altered morphology was assessed by defining altered mitochondria as having a fragmented crista, inhomogeneous electron transparent matrix, and/or U-, C-, or ring-shaped morphology (O). P–U, Triclosan alters fusion and fission protein levels. Dynamin-related protein 1 (Drp1) and optic atrophy 1 (Opa1) protein levels were analyzed via ProteinSimple Wes Immunoassay system. Virtual blot view of Wes results showing the Drp1 and Opa1 levels obtained in dLN lysates after 4days of triclosan exposure (P). Area under the curve (AUC) analysis of Drp1 (Q) and Opa1 (R) with normalization to total protein. Virtual blot view of Wes results showing the Drp1 and Opa1 levels obtained in skin tissue lysates after 4days of triclosan exposure (S). AUC analysis of Drp1 (T) and Opa1 (U) with normalization to total protein. Data shown as the mean (± SEM) of 3–5 mice per group. Statistical significance relative to 0% vehicle control determined by one-way ANOVA followed by Dunnett’s post-test indicated as **p<.01, ***p<.001.

Article Snippet: Total protein was used as a loading control and for normalization. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Reference Sample Concentration Antibody Dilution Secondary Antibody Opa1 Novus Biologicals 0.2 mg/ml (skin) 1:200 (skin) Anti-rabbit-HRP (Wes detection kit) NB-110-55290SS 0.2 mg/ml (dLN) 1:200 (dLN) Drp1 Novus Biologicals 0.1 mg/ml (skin) 1:150 (skin) NB110-55288SS 0.1 mg/ml (dLN) 1:150 (dLN) Open in a separate window Primary and Secondary Antibodies Used for ProteinSimple Wes Immunoassay

Techniques: Transmission Assay, Electron Microscopy, Control

Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Topical Application of the Antimicrobial Agent Triclosan Induces NLRP3 Inflammasome Activation and Mitochondrial Dysfunction

doi: 10.1093/toxsci/kfaa056

Figure Lengend Snippet: Schematic of the effects of dermal triclosan exposure on the skin and draining lymph node (dLN) in a BALB/c mouse model. Dermal triclosan exposure increases S100a8 gene expression in the skin causing toll-like receptor signaling activation leading to an increase in Nlrp3 and pro-Il-1b gene expression (priming signal). An increase in extracellular ATP occurs in the skin upon triclosan exposure, possibly acting as a secondary signal leading to caspase-1 activation. Active caspase-1 then cleaves pro-IL-1β leading to an increase in its active form. The activation of caspase-1 and subsequent cleavage of IL-1β could be responsible for the increase in myeloid and inflammatory monocyte cells migrating into the skin. Caspase-1 activation also occurs in the dLN with an increase in mature IL-1β secretion. Myeloid cells, neutrophils, and macrophages increase in number in the dLN, possibly migrating due to the caspase-1 activation. Triclosan also caused a decrease in mitochondrial mass, mitochondrial ROS, and mitochondrial membrane potential in the dLN with an increase in altered mitochondria. Upward facing arrows indicate an increase found in this study and the downward facing arrows indicate a decrease. Dotted arrows/lines are effects suggested in the literature. Abbreviations: Drp1, dynamin-related protein 1; Opa1, optic atrophy 1.

Article Snippet: Total protein was used as a loading control and for normalization. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Reference Sample Concentration Antibody Dilution Secondary Antibody Opa1 Novus Biologicals 0.2 mg/ml (skin) 1:200 (skin) Anti-rabbit-HRP (Wes detection kit) NB-110-55290SS 0.2 mg/ml (dLN) 1:200 (dLN) Drp1 Novus Biologicals 0.1 mg/ml (skin) 1:150 (skin) NB110-55288SS 0.1 mg/ml (dLN) 1:150 (dLN) Open in a separate window Primary and Secondary Antibodies Used for ProteinSimple Wes Immunoassay

Techniques: Expressing, Activation Assay, Membrane

Journal: Cells

Article Title: A Combination Therapy of Urolithin A+EGCG Has Stronger Protective Effects than Single Drug Urolithin A in a Humanized Amyloid Beta Knockin Mice for Late-Onset Alzheimer’s Disease

doi: 10.3390/cells11172660

Figure Lengend Snippet: Summary of q RT-PCR oligonucleotide primers used in measuring mRNA expression in mitochondrial structural, biogenesis, synaptic genes and autophagy and mitophagy genes.

Article Snippet: OPA1 Rabbit polyclonal (#NBP2-59770) 1:100 , Novus Biological, Littleton, CO , Donkey anti-rabbit HRP 1:200 , GE Healthcare Amersham, Piscataway, NJ.

Techniques: Expressing, Sequencing

Journal: Cells

Article Title: A Combination Therapy of Urolithin A+EGCG Has Stronger Protective Effects than Single Drug Urolithin A in a Humanized Amyloid Beta Knockin Mice for Late-Onset Alzheimer’s Disease

doi: 10.3390/cells11172660

Figure Lengend Snippet: Summary of antibody dilutions and conditions used in the immunofluorescence analysis of mitochondrial dynamics, mitochondrial biogenesis, synaptic mitophagy and autophagy proteins in mitophagy enhancer-treated and -untreated hAbKI mice.

Article Snippet: OPA1 Rabbit polyclonal (#NBP2-59770) 1:100 , Novus Biological, Littleton, CO , Donkey anti-rabbit HRP 1:200 , GE Healthcare Amersham, Piscataway, NJ.

Techniques: Immunofluorescence

Journal: Cells

Article Title: A Combination Therapy of Urolithin A+EGCG Has Stronger Protective Effects than Single Drug Urolithin A in a Humanized Amyloid Beta Knockin Mice for Late-Onset Alzheimer’s Disease

doi: 10.3390/cells11172660

Figure Lengend Snippet: Summary of antibody dilutions and conditions used in the immunoblotting analysis of mitochondrial dynamics, mitochondrial biogenesis, synaptic mitophagy and autophagy proteins in mitophagy enhancer-treated and -untreated hAbKI mice.

Article Snippet: OPA1 Rabbit polyclonal (#NBP2-59770) 1:100 , Novus Biological, Littleton, CO , Donkey anti-rabbit HRP 1:200 , GE Healthcare Amersham, Piscataway, NJ.

Techniques: Western Blot

Journal: Cells

Article Title: A Combination Therapy of Urolithin A+EGCG Has Stronger Protective Effects than Single Drug Urolithin A in a Humanized Amyloid Beta Knockin Mice for Late-Onset Alzheimer’s Disease

doi: 10.3390/cells11172660

Figure Lengend Snippet: Summary of mRNA fold changes comparison in Urolithin A-treated hAbKI mice and Urolithin A+EGCG treated hAbKI mice.

Article Snippet: OPA1 Rabbit polyclonal (#NBP2-59770) 1:100 , Novus Biological, Littleton, CO , Donkey anti-rabbit HRP 1:200 , GE Healthcare Amersham, Piscataway, NJ.

Techniques: Comparison

Journal: Cells

Article Title: A Combination Therapy of Urolithin A+EGCG Has Stronger Protective Effects than Single Drug Urolithin A in a Humanized Amyloid Beta Knockin Mice for Late-Onset Alzheimer’s Disease

doi: 10.3390/cells11172660

Figure Lengend Snippet: Immunoblotting analysis of mitochondrial dynamic proteins . Immunoblotting analysis was assessed using lysates prepared from post-mortem brains of seven-month-old hAbKI mice and treated hAbKI mice with Uralithin A and EGCG. ( A ) Representative immunoblots for hAbKI mice and treated hAbKI mice with Urolithin A and EGCG. ( B ) Quantitative-densitometry analysis for mitochondrial fission genes Drp1 and Fis1 and fusion proteins, which were significantly decreased in the treated hAbKI mice Urolithin A and EGCG as compared to the hAbKI mice. Mitochondrial fusion proteins Mfn1, Mfn2 and Opa1 were significantly increased in urolithin A-treated hAbKI mice and combined treatment of urolithin A+EGCG in 7-month-old hAbKI mice.

Article Snippet: OPA1 Rabbit polyclonal (#NBP2-59770) 1:100 , Novus Biological, Littleton, CO , Donkey anti-rabbit HRP 1:200 , GE Healthcare Amersham, Piscataway, NJ.

Techniques: Western Blot

Journal: Experimental & Molecular Medicine

Article Title: Astrocyte–neuron crosstalk through extracellular vesicle-shuttled miRNA-382-5p promotes traumatic brain injury

doi: 10.1038/s12276-024-01355-3

Figure Lengend Snippet: a Overlap of potential targets of miRNA-382-5p predicted by the TargetScan, miRWalk, miRDB, RNAhybrid, and miRanda databases. b Venn diagram of putative target genes of miRNA-382-5p overlapping with mitochondria-related genes. c Wild-type and mutant OPA1 3′-UTR reporter constructs. d Luciferase reporter assay results for HEK293T cells cotransfected with the indicated wild-type or mutant 3’-UTR constructs and the miRNA-382-5p mimic ( n = 6 per group; one-way ANOVA). e , f WB analysis and densitometric quantification of OPA1 levels in primary neurons transfected with or without the miRNA-382-5p mimic ( n = 6 per group; Student’s t test). g MitoTracker was used to label mitochondria in primary neurons, mitochondrial morphology was examined via fluorescence microscopy, and mitochondrial morphological features were quantified (aspect ratios) using ImageJ software. h The boxed area next to each micrograph shows an magnified version of the white square ( n = 6 per group, one-way ANOVA). i , j Representative images of MitoSOX fluorescence and quantitative comparison of mitochondria-derived ROS levels ( n = 6 per group; one-way ANOVA). k , l Representative images of fluorescence staining and quantitative comparison of JC-1 aggregates (red fluorescence) and JC-1 monomers (green fluorescence) in cultured primary neurons from different experimental groups ( n = 6 per group; one-way ANOVA). The data are presented as the means ± SDs; ** p < 0.01, *** p < 0.001, and NS not significant.

Article Snippet: Neuron-specific OPA1 knockout mice were generated by crossing Opa1 f/f mice (Cyagen, USA, Inc., CA, USA) with Map2-CreERT2 mice (Shanghai Model Center) and then treating the offspring with 75 mg/kg tamoxifen (#S1238, Selleck) for 5 days.

Techniques: Mutagenesis, Construct, Luciferase, Reporter Assay, Transfection, Fluorescence, Microscopy, Software, Comparison, Derivative Assay, Staining, Cell Culture

Journal: Experimental & Molecular Medicine

Article Title: Astrocyte–neuron crosstalk through extracellular vesicle-shuttled miRNA-382-5p promotes traumatic brain injury

doi: 10.1038/s12276-024-01355-3

Figure Lengend Snippet: a Cartoon illustrating the process used to construct OPA1 conditional knockout mice. b Results showing the identification of the neuron-specific OPA1 conditional knockout mice. c TEM images showing cortical mitochondrial crista remodeling in the Opa1 f/f group or Opa1 CKO group subjected to sham surgery or TBI. d Fifty randomly selected mitochondria per sample were scored in three categories ( n = 6 per group, one-way ANOVA): more than four cristae (Class I), between two and three cristae (Class II), and one or no cristae (Class III) per mitochondrion. e Fifty mitochondria per sample were scored according to matrix density and swelling ( n = 6 per group, one-way ANOVA): dense matrix (Class A) and swollen mitochondria with a hypodense matrix (Class B). f The length of the mitochondria in each group, which is represented by the mean length of 50 randomly selected mitochondria ( n = 6 per group; one-way ANOVA). g , h Representative MR images and statistical analysis of histological impairments at 24 h after TBI ( n = 6 per group; Student’s t test). i Statistical analysis of brain edema. n = 6 per group; Student’ s t test) j – l The neurological function deficit scores of the Opa1 f/f group or Opa1 CKO group ( n = 12 per group, two-way ANOVA). The data are presented as the means ± SDs; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Neuron-specific OPA1 knockout mice were generated by crossing Opa1 f/f mice (Cyagen, USA, Inc., CA, USA) with Map2-CreERT2 mice (Shanghai Model Center) and then treating the offspring with 75 mg/kg tamoxifen (#S1238, Selleck) for 5 days.

Techniques: Construct, Knock-Out

Journal: Experimental & Molecular Medicine

Article Title: Astrocyte–neuron crosstalk through extracellular vesicle-shuttled miRNA-382-5p promotes traumatic brain injury

doi: 10.1038/s12276-024-01355-3

Figure Lengend Snippet: a , b WB analysis and densitometric quantification of the levels of the OPA1 protein in the perilesional cortex of mice injected with or without RVG-miR-382-5pi-EVs ( n = 6 per group; one-way ANOVA). c TEM images showing that the loss of mitochondrial cristae in the perilesional cortex was inhibited in the RVG-NC-EVs group and the RVG-miR-382-5pi-EVs group. d Fifty mitochondria per sample were assigned to three categories: more than four cristae (Class I), between two and three cristae (Class II), and one or no cristae (Class III) per mitochondrion ( n = 6 per group, one-way ANOVA). e Fifty mitochondria per sample were scored according to matrix density and swelling: dense matrix (Class A) and swollen mitochondria with a hypodense matrix (Class B) ( n = 6 per group, one-way ANOVA). f Representative quantitative results of the length of 50 mitochondria per experiment ( n = 6 per group; one-way ANOVA). g , h Representative MR images and statistical analysis of histological impairments at 24 h after TBI ( n = 6 per group; one-way ANOVA). i Statistical analysis of brain edema ( n = 6 per group, one-way ANOVA). j‒l Neurological function deficit scores of the RVG-NC-EVs group and RVG-miR-382-5pi-EVs group ( n = 12 per group, two-way ANOVA). The data are presented as the means ± SDs; * p < 0.05, ** p < 0.01, *** p < 0.001, and NS not significant.

Article Snippet: Neuron-specific OPA1 knockout mice were generated by crossing Opa1 f/f mice (Cyagen, USA, Inc., CA, USA) with Map2-CreERT2 mice (Shanghai Model Center) and then treating the offspring with 75 mg/kg tamoxifen (#S1238, Selleck) for 5 days.

Techniques: Injection

Journal: Scientific Reports

Article Title: Mitofusion is required for MOTS‐c induced GLUT4 translocation

doi: 10.1038/s41598-021-93735-2

Figure Lengend Snippet: Treatment of U-2 OS cells with MOTS-c initiated mitochondrial fusion. Treatment with MOTS-c increased MfN2 and OPA1 protein expression in ( A , B ) whole cells and ( C , D ) mitochondria. U-2 OS cells grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) plus 1% pen-strep were treated with vehicle (Nuclease-Free Water) or MOTS-c (25, 50 or 100 μM) for 48 h and western blot was performed to quantify the protein expression level of selected biomarkers. Mitochondria were extracted from cells treated with 100 μM MOTS-c only. GAPDH was used as the internal control for whole cell analysis while TOMM20 was used as the mitochondrial control. All full-length blots are presented in Supplementary Figure . Data are means ± SEM of three to four experiments. Statistical analysis was conducted using ordinary one-way ANOVA followed by Dunnett's multiple comparisons test vs vehicle; results are presented as *p < 0.05, and **p < 0.01 vs. vehicle.

Article Snippet: The OPA1 (NBP2-59770SS) antibody was obtained from Novus Biologicals (Toronto, Canada).

Techniques: Expressing, Modification, Western Blot, Control, Cell Analysis