Journal: NPJ Parkinson's Disease

Article Title: Protection from α-Synuclein induced dopaminergic neurodegeneration by overexpression of the mitochondrial import receptor TOM20

doi: 10.1038/s41531-020-00139-6

Figure Lengend Snippet: AAV2 vectors driving protein overexpression were injected via stereotaxic surgery into the right substantia nigra of adult, male Lewis rats. Vector combinations of AAV2-TOM20/GFP, AAV2-αSyn/GFP, and AAV2-TOM20/αSyn represent the three treatment groups utilized within this study ( a ). Following vector infusion, animals were monitored daily for motor behavior, and euthanized for tissue collection 12-weeks post-injection ( b ). Representative images from each vector treatment group comparing ipsilateral (injected) and contralateral (non-injected) brain hemispheres; TOM20 (red), αSyn (cyan), GFP (green); scale bar 100 μm ( c ). Target protein overexpression in neurons 12 weeks following vector expression; TOM20 (red), αSyn (white), GFP (green; d) .

Article Snippet: TOM20 (Ms) , 612278 , 1:500 , , BD Biosciences (San Jose, CA).

Techniques: Over Expression, Injection, Plasmid Preparation, Expressing

Journal: NPJ Parkinson's Disease

Article Title: Protection from α-Synuclein induced dopaminergic neurodegeneration by overexpression of the mitochondrial import receptor TOM20

doi: 10.1038/s41531-020-00139-6

Figure Lengend Snippet: Human αSyn protein expression was detectable within the injected hemisphere of AAV2-αSyn/GFP and AAV2-TOM20/αSyn and was expressed at similar levels in both treatment groups receiving the human αSyn vector; (F(2, 26 = 4.6), p = 0.0192, ANOVA); scale bar 100 μm ( a , b ). Insoluble αSyn (proteinase K resistant) was confirmed in animals injected with AAV2-αSyn vectors; representative image from AAV2-TOM20/αSyn; scale bar 100 μm ( c ). AAV2-TOM20 vector infusion resulted in significantly elevated levels of TOM20 within neurons of the ventral midbrain, which was not affected by co-expression of AAV2-αSyn; (F(2, 6 = 17.5), p = 0.0031, ANOVA; d , e ). Error bars depict standard error of the mean (s.e.m.).

Article Snippet: TOM20 (Ms) , 612278 , 1:500 , , BD Biosciences (San Jose, CA).

Techniques: Expressing, Injection, Plasmid Preparation

Journal: NPJ Parkinson's Disease

Article Title: Protection from α-Synuclein induced dopaminergic neurodegeneration by overexpression of the mitochondrial import receptor TOM20

doi: 10.1038/s41531-020-00139-6

Figure Lengend Snippet: Dopaminergic terminals within the striatum (tyrosine hydroxylase; TH; red) were significantly reduced compared to the contralateral side in animals expressing AAV2-αSyn/GFP, but not in animals expressing AAV2-TOM20/αSyn; TH (red), αSyn (cyan), GFP (green); (F(5, 95 = 3.0), p = 0.0146, ANOVA; a , b ). Representative images from the ipsilateral and contralateral hemispheres of each treatment group show significant dopaminergic neuron loss (TH, red) following AAV2-αSyn/GFP treatment, which was reduced by co-expression with TOM20 (AAV2-TOM20/αSyn); (F(5, 20 = 12.07), p < 0.0001, ANOVA); scale bars 100 μm ( c , d ). Error bars depict standard error of the mean (s.e.m.).

Article Snippet: TOM20 (Ms) , 612278 , 1:500 , , BD Biosciences (San Jose, CA).

Techniques: Expressing

Journal: NPJ Parkinson's Disease

Article Title: Protection from α-Synuclein induced dopaminergic neurodegeneration by overexpression of the mitochondrial import receptor TOM20

doi: 10.1038/s41531-020-00139-6

Figure Lengend Snippet: NADH:Ubiquinone Oxidoreductase Core Subunit S3 (NDUFS3, white) is a component of complex I of the electron transport chain. NDUFS3 expression is significantly reduced in dopaminergic neurons of animals expressing AAV2-αSyn/GFP, and rescued by AAV2-TOM20 co-expression; TOM20 (red), GFP (green), TH (blue); (F(2, 10 = 11.82), p = 0.0023, ANOVA; ( a , b ). Succinate dehydrogenase complex flavoprotein subunit A (SDHA, yellow) encodes the catalytic subunit of succinate-ubiquinone oxidoreductase of the electron transport chain, was reduced within mitochondria of dopaminergic neurons following αSyn overexpression, and rescued by AAV2-TOM20 co-expression; TOM20 (white), TH (magenta); (F(2, 23 = 14.57), p = 0.0001, ANOVA; c , d ). Cytochrome c oxidase or complex IV (COX IV, red) the final enzyme in the mitochondrial transport chain, displayed a significant reduction within mitochondria in dopaminergic neurons expressing AAV2-αSyn/GFP, but not neurons expressing AAV2-TOM20/αSyn; TOM20 (red), TH (green); (F(2, 23 = 8.46), p = 0.0018, ANOVA; e , f ). Error bars depict standard error of the mean (s.e.m.).

Article Snippet: TOM20 (Ms) , 612278 , 1:500 , , BD Biosciences (San Jose, CA).

Techniques: Expressing, Over Expression

Journal: NPJ Parkinson's Disease

Article Title: Protection from α-Synuclein induced dopaminergic neurodegeneration by overexpression of the mitochondrial import receptor TOM20

doi: 10.1038/s41531-020-00139-6

Figure Lengend Snippet: The redox-sensitive mitochondrial chaperone protein GRP75 was significantly elevated in dopaminergic neurons expressing AAV2-TOM20/αSyn, but not AAV2-αSyn/GFP, or AAV2-TOM20/GFP; (F(2, 4 = 167.2), p = 0.0001, ANOVA; a , b ). Acute rotenone treatment causes the endogenous accumulation of αSyn (5-day ROT) that corresponded to a significant elevation in GRP75 expression within mitochondria, which was diminished in chronic/endpoint rotenone treated rats; (F(2, 15 = 14.69) p = 0.0003, ANOVA; c , d ). Total TOM20 protein expression in the dopaminergic neurons of the SN was not significantly changed following rotenone treatment ( e ). Error bars depict standard error of the mean (s.e.m.).

Article Snippet: TOM20 (Ms) , 612278 , 1:500 , , BD Biosciences (San Jose, CA).

Techniques: Expressing

Journal: NPJ Parkinson's Disease

Article Title: Protection from α-Synuclein induced dopaminergic neurodegeneration by overexpression of the mitochondrial import receptor TOM20

doi: 10.1038/s41531-020-00139-6

Figure Lengend Snippet: Vector information.

Article Snippet: TOM20 (Ms) , 612278 , 1:500 , , BD Biosciences (San Jose, CA).

Techniques: Plasmid Preparation, Expressing, Injection

Journal: NPJ Parkinson's Disease

Article Title: Protection from α-Synuclein induced dopaminergic neurodegeneration by overexpression of the mitochondrial import receptor TOM20

doi: 10.1038/s41531-020-00139-6

Figure Lengend Snippet: Antibody information.

Article Snippet: TOM20 (Ms) , 612278 , 1:500 , , BD Biosciences (San Jose, CA).

Techniques: Concentration Assay

Journal: GeroScience

Article Title: Unbiased proteomic analysis of extracellular vesicles secreted by senescent human vascular smooth muscle cells reveals their ability to modulate immune cell functions

doi: 10.1007/s11357-022-00625-0

Figure Lengend Snippet: Characterization of extracellular vesicles secreted by VSMCs. a Isolated EVs, obtained after ultracentrifugation of conditioned medium, observed in TEM; b Western blot analysis of common exosome markers (CD81, CD63, and Flotilin-1) in EV fraction (20µg of each sample). The purity of isolated EVs was confirmed by lack of expression of Golgi (GM130) and mitochondrial (TOM20) markers, TLC — total cell lysate; c Size measurement of EVs using NanoSight nanoparticle tracking analysis (NTA). d The total number of EVs secreted by young and senescent cells measured by NTA; EVs from at least 9 independent isolations from each experimental conditions, control, SIPS and RS, cells were analyzed. e Comparison of the number of CD63 positive EVs secreted by VSMCs (ExoElisa); EVs from at least 9 independent isolations were analyzed. f Representative blots presenting the differences in the level of CD81, CD63, and Flotilin-1 protein in EVs secreted by 2 × 10 5 young and senescent cells

Article Snippet: The primary antibodies used were anti-Flotilin-1 (BD Transduction Laboratories, 1:500), anti-CD63 (Abcam 1:1000), anti-CD81 (Abcam, 1:500), anti-GM130 (Cell Signaling 1:1000), and anti-TOM20 (GeneTex, 1:500).

Techniques: Isolation, Western Blot, Expressing

Journal: PLOS One

Article Title: Characterizing mitochondrial phenotypes and MERCS in aged human skeletal muscle myoblasts

doi: 10.1371/journal.pone.0343604

Figure Lengend Snippet: (a) Results of oxygen consumption measurements using the Seahorse Flux Analyzer. Each point represents the mean ± S. D. deviation for young and old cells (n = 3 biological replicates), with 20,000 cells per measurement. (b) Results of mitochondrial morphology. The images are partial views of cells stained with TOM20 antibody, with the scale bar representing 10 μm. The graph shows the average mitochondrial size per cell measured from the images, presented as violin plots (n = 3 biological replicates; cells counted: young, 239, young+FCCP, 220, old, 264). p -values were calculated using Dunnett’s test with young as the control, **; p < 0.01, ***; p < 0.001. (c) Results of mitochondrial DNA quantification (n = 3 biological replicates; cells counted: young, 3, old, 3). The quantity of mitochondrial genes was normalized using bAct as an internal standard. q-values were calculated using Welch’s t-test for multiple comparisons.

Article Snippet: The cells were then permeabilized for 10 min with 0.2% Triton and blocked by incubation with Blocking One Histo (Nacalai) for 1 h. After blocking, the cells were incubated for 1 h at RT with mouse monoclonal anti-TOM20 antibody (ab56783; 1:40 in PBS-T with 5% Blocking One Histo).

Techniques: Staining, Control

Journal: Theranostics

Article Title: Alisol A ameliorates vascular cognitive impairment via AMPK/NAMPT/SIRT1-mediated regulation of cholesterol and autophagy

doi: 10.7150/thno.112661

Figure Lengend Snippet: Alisol A activates the NAMPT-related signaling pathway, thereby facilitating lysine deacetylation and autophagy. (A) Schematic. (B) Total proteins were collected from the mice hippocampal tissues and evaluated by WB, including AMPK, p-AMPKα Thr172 or Thr183 , NAMPT, SIRT1, PGC1-α, and AcetyI-Lysine, bar graph represents semi-quantification of WB (C) , n = 3; (D) NAD + levels in the hippocampus of Ldlr -/- mice were determined by the NAD + Assay Kit, n = 8; (E) NAD⁺ content in HT22 cells or in primary neurons was measured using an NAD assay kit, n = 8; (F) WB analysis of AMPK, p-AMPKα Thr172 or Thr183 , NAMPT, SIRT1, PGC1-α, and AcetyI-Lysine in cells, and quantification result was normalized against the levels of GAPDH, n = 3; (G) Representative images of NAMPT (green) and SIRT1 (red) double immunofluorescence staining, scale: 5 μm; and quantification of immunofluorescence stain intensity using Image J; (H) WB analysis of P62, BECLIN1, PARKIN, PINK1, COXIV, TOM20, and LC3B in cells, and quantification result was normalized against the levels of GAPDH or COXIV, n = 3; (I) Changes in MMP was monitored by staining with JC1, scale: 100 μm; and quantification of the percentage of MMP, n = 8. Compared to the 5, * P < 0.05, ** P < 0.01; compared to the 6, # P < 0.05, ## P < 0.01. Compared to the control group, * P < 0.05, ** P < 0.01; compared to the Ldlr -/- , # P < 0.05, ## P < 0.01.

Article Snippet: Anti-GFAP antibody (GB12096, Servicebio, 1:500), anti-IBA-1 antibody (GB12105, Servicebio, 1:500). anti-PSD95 antibody (20665-1-AP, Proteintech, 1:300), anti-SYNAPSIN-1 antibody (20258-1-AP, Proteintech, 1:300), anti-UCP2 antibody (GB11377, Servicebio, 1:300), anti-CD68 antibody ( GB113109 , Servicebio, 1:300), anti-α-SMA antibody ( GB111364 , Servicebio, 1:300), anti-HMGCR antibody (DF6518, Affinity, 1:200), anti-ABCA1 Polyclonal antibody (26564-1-AP, Proteintech, 1:300). anti-NAMPT/PBEF Polyclonal antibody (11776-1-AP, Proteintech, 1:300), anti-SIRT1 antibody (13161-1-AP, Proteintech, 1:300). anti-CYP46A1 Polyclonal antibody (124861-1-AP, Proteintech, 1:300). anti-OPTN antibody ( GB114324 , Servicebio, 1:300), anti-TOM20 antibody ( GB111481 , Servicebio, 1:300), anti-NMDAR2A/GRIN2A Polyclonal antibody (28525-1-AP, Proteintech, 1:300), anti-NMDA2B antibody (21920-1-AP, Proteintech, 1:300).

Techniques: Double Immunofluorescence Staining, Immunofluorescence, Staining, Control

Journal: Theranostics

Article Title: Alisol A ameliorates vascular cognitive impairment via AMPK/NAMPT/SIRT1-mediated regulation of cholesterol and autophagy

doi: 10.7150/thno.112661

Figure Lengend Snippet: Modulation of NAMPT activity and its network by Alisol A restores cholesterol balance, mitophagy, and synaptic function in Ldlr -/- mice. (A) WB analysis of P62, BECLIN1, PARKIN, PINK1, COXIV, TOM20, and LC3B protein quantitation for WB, n = 3. (B) WB analysis of P62, BECLIN1, PARKIN, PINK1, COXIV, TOM20, and LC3B in cells, and quantification result was normalized against the levels of GAPDH or COXIV, n = 3; (C) Changes in MMP was monitored by staining with JC1 in HT22 cells, scale: 100 μm; The ratio of red/green fluorescence intenity was employed to assess MMP, n = 8; (D) Representative IF images of OPTN and TOM20 in HT22 cells, scale: 2 μm; Quantification of IF stain intensity using Image J, n = 8; IF and colocalization analysis. (E) WB analysis of PINK1 in HT22 cells and quantification result was normalized against the levels of GAPDH, n = 3; (F) Subsequent COIP experiments employed NAMPT antibodies to assess interactions with NAMPT and PINK1, using IgG as a control. (G) Illustrative IF images depicting hippocampal neurons located within the CA3 subregion, scale: 100 μm, enlarge scale: 10 μm, n = 5; IF was quantified for UCP2 (red); (H) The phenomenon of intercellular ROS generation was observed through the detection of intracellular ROS levels using the DCFH-DA probe following different treatment protocols, scale: 100 μm, n = 5; (I) Intracellular ROS levels were measured using flow cytometry, comparative analysis of fluorescence intensity within peak plots derived from flow cytometry, n = 5; (J) Synapses in the hippocampal CA1 and DG region were revealed by IF staining for the pre-and postsynaptic markers, Synapsin-1 (red) and PSD95 (green) respectively, scale: 10 μm, enlarge scale: 2 μm, n = 5; and quantification of IF; (K) Golgi-Cox Staining Hippocampal Golgi-Cox staining was performed as previously described, scale: 100 μm, enlarge scale: 20 μm, n = 8; Track the neural branches after Golgi staining and analysis of Sholl results and statistical data; Analysis of dendritic spine density in neurons stained with Golgi, along with the quantification of mushroom spines, scale: 2 μm; (L) Representative IF images of NMDA 2A (green) and NMDA 2B (red), scale: 2 μm; and quantification of IF, n = 5. Compared to the Ldlr -/- mice , * P < 0.05, ** P < 0.01; compared to the FK866 group, # P < 0.05, ## P < 0.01. Compared to the 1, * P < 0.05, ** P < 0.01; compared to the 3, # P < 0.05, ## P < 0.01. DMSO: HT22 +DMSO; Rosup: HT22 + Aβ 25-35 (20 μM) and complex (50 μg/ml of cholesterol) +H 2 O 2 0.1 mM ; CCCP: HT22+ CCCP 10 μM. Compared to the 5, * P < 0.05, ** P < 0.01; compared to the 7, # P < 0.05, ## P < 0.01.

Article Snippet: Anti-GFAP antibody (GB12096, Servicebio, 1:500), anti-IBA-1 antibody (GB12105, Servicebio, 1:500). anti-PSD95 antibody (20665-1-AP, Proteintech, 1:300), anti-SYNAPSIN-1 antibody (20258-1-AP, Proteintech, 1:300), anti-UCP2 antibody (GB11377, Servicebio, 1:300), anti-CD68 antibody ( GB113109 , Servicebio, 1:300), anti-α-SMA antibody ( GB111364 , Servicebio, 1:300), anti-HMGCR antibody (DF6518, Affinity, 1:200), anti-ABCA1 Polyclonal antibody (26564-1-AP, Proteintech, 1:300). anti-NAMPT/PBEF Polyclonal antibody (11776-1-AP, Proteintech, 1:300), anti-SIRT1 antibody (13161-1-AP, Proteintech, 1:300). anti-CYP46A1 Polyclonal antibody (124861-1-AP, Proteintech, 1:300). anti-OPTN antibody ( GB114324 , Servicebio, 1:300), anti-TOM20 antibody ( GB111481 , Servicebio, 1:300), anti-NMDAR2A/GRIN2A Polyclonal antibody (28525-1-AP, Proteintech, 1:300), anti-NMDA2B antibody (21920-1-AP, Proteintech, 1:300).

Techniques: Activity Assay, Protein Quantitation, Staining, Fluorescence, Control, Flow Cytometry, Derivative Assay