Journal: Biomedicines

Article Title: The Imbalance of Astrocytic Mitochondrial Dynamics Following Blast-Induced Traumatic Brain Injury.

doi: 10.3390/biomedicines11020329

Figure Lengend Snippet: Figure 3. Mitochondrial Network Analysis (MiNA) software descriptors from astrocyte mitochondrial morphology post single mechanical exposure—in vitro model of bTBI. (a) Representative images (63x) of TOMM20 and skeletonized for data acquisition at 4 hours post single mechanical exposure. Astrocytes presented a significant increase in the number of individuals fragmented mitochondria (puncta and rod) and a small number of networks. (b) Representative images of TOMM20 and skeletonized for data acquisition at 24 h post single mechanical exposure. Astrocytes still presented a significant increase in the number of individuals fragmented mitochondria (puncta and rod), displayed

Article Snippet: Fixed cells were incubated, for three hours at 4°C, in blocking buffer with primary antibody TOMM20 at 1:100 dilution (Cat#: NBP181556; Novus Biologicals, Englewood, CO, USA) and GFAP at 1:100 dilution (Cat#: 13-0300; ThermoFisher, Waltham, MA, USA), this step was carefully conducted by placing the cov- Biomedicines 2023, 11, 329 7 of 21 erslip with upturned cells into a humidified chamber.

Techniques: Software, In Vitro

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A Chemically Defined, Xeno- and Blood-Free Culture Medium Sustains Increased Production of Small Extracellular Vesicles From Mesenchymal Stem Cells

doi: 10.3389/fbioe.2021.619930

Figure Lengend Snippet: Characterization of isolated UC-MSC-derived sEV produced in DMEM, Oxium TM EXO, and commercial medium. The sEV isolated from the different conditioned media were evaluated in terms of particle concentration, size, classical surface/interior markers, and morphology. (A) Histogram showing the particles’ concentrations according to their size. Violet line = DMEM-derived sEV; orange line = Oxium TM EXO-derived sEV; light blue line = commercial medium-derived sEV. The mean concentration obtained through NTA of five videos for each type of sEV is shown. (B) Size’s mean and mode obtained for each type of sEV. (C) Size’s mode (left panel) and standard deviation data (right panel) dispersion. (D) Percentage distribution of isolated particles’ concentrations according to their size: 0–50 nm, 51–200 nm, 201–300 nm, and >301 nm. (E) Representative histograms of median fluorescence intensity (MFI) obtained by flow cytometry of classical sEV surface markers. Gray = isotype control; violet = DMEM; orange = Oxium TM EXO, light blue = commercial medium. (F) Western blot, illustrating the presence of the sEV’s membrane-associated protein Flotillin-1 and the sEV’s luminal-scaffold protein Syntenin-1 (involved in sEV’s biogenesis). Note that in the isolated sEV there is minimal or no detectable contamination by Calnexin (endoplasmic reticulum) or TOMM20 (mitochondria), respectively. (G) Transmission electron microscopy (TEM) by uranyl acetate negative staining of isolated sEV from ultracentrifuge. The graphs show mean ± SEM. n = 2.

Article Snippet: Primary antibodies used were Syntenin-1 (1:1000; Novus Biologicals, Centennial, CO, United States, Cat. #NBP2-76873), Flotillin-1 (1:2000; Abcam Inc., Cambridge, MA, United States, Cat. #ab133497), Calnexin (1:2,000; Abcam Inc., Cambridge, MA, United States Cat. #ab22595), and TOMM20 (1:1,000; Novus Biologicals, Centennial, CO, United States, Cat. #NBP2-67501).

Techniques: Isolation, Derivative Assay, Produced, Concentration Assay, Standard Deviation, Dispersion, Fluorescence, Flow Cytometry, Control, Western Blot, Membrane, Transmission Assay, Electron Microscopy, Negative Staining

Journal: BMC Research Notes

Article Title: Spatial profiles of markers of glycolysis, mitochondria, and proton pumps in a rat glioma suggest coordinated programming for proliferation

doi: 10.1186/s13104-015-1191-z

Figure Lengend Snippet: Upregulation of GAPDH and Tom20 in C6 gliomas. a A section of rat brain stained with hematoxilin and eosin to show a glioma produced by implantation of C6 cells in the right caudate nucleus. In this case, cells also grew in the region of the syringe needle track through the cortex. ( b – d ). A small glioma labeled by bisbenzamide ( b ) and with antibodies against Tom20 ( c ) and GAPDH ( d ). The brightness and contrast have been increased. e Illustration of a strip perpendicular to the rim of a glioma (labeled with bisbenzamide) and along which intensity profiles were measured. f , g Tom20 and GAPDH fluorescence along such a strip (barely visible without enhancement). The graphs show the raw intensity values given by ImageJ.

Article Snippet: The antibody for Tom20 was a mouse monoclonal (Novus Biologicals H00009804_M01; called TOMM20) raised against a 146 AA recombinant protein, and used at 1/500.

Techniques: Staining, Produced, Labeling, Stripping Membranes, Fluorescence

Journal: BMC Research Notes

Article Title: Spatial profiles of markers of glycolysis, mitochondria, and proton pumps in a rat glioma suggest coordinated programming for proliferation

doi: 10.1186/s13104-015-1191-z

Figure Lengend Snippet: Averaged profiles of upreglation of Tom20 and GAPDH. a Means of 16 pairs of profiles for Tom20 and GAPDH. b The mean ratios of individual pairs of profiles, Tom20/GAPDH. The ratio is greatest in the range 0.4–0.8 mm ( vertical lines ). c Mean values for individual profiles over the distance 0.4–0.8 mm into the glioma.

Article Snippet: The antibody for Tom20 was a mouse monoclonal (Novus Biologicals H00009804_M01; called TOMM20) raised against a 146 AA recombinant protein, and used at 1/500.

Techniques:

Journal: Oncotarget

Article Title: Hypoxia-mediated alterations and their role in the HER-2/neuregulated CREB status and localization

doi: 10.18632/oncotarget.10474

Figure Lengend Snippet: A. The distribution of CREB in different cell fractions was analysed by using a commercial kit for separations followed by Western blot analysis of the distinct fractions. Anti-MEK1 (cytosol) and anti-histone H3 (nucleus) mAb served as markers for the subcellular compartments. “W” marks the whole cell lysate, “C” the cytosolic fraction, “M” the membrane/mitochondrial fraction and “N” the nuclear fraction. The picture represents one of three independent experiments. B. Cells were incubated for 24 h under hypoxia or normoxia and 30 min before terminating the cells were stained with 500 nM MitoTracker. After removing the media and fixation with 4% PFA for 20 min the cells were permeabilized with 0.5% Triton X100 in HBBS (HBSS-T) for 30 min. Following incubation with CREB antibody overnight at 4°C the cells were washed three times with HBSS, incubated with the secondary antibody (rabbit-Alexa 488) for 1 h, washed again three times with HBSS and then the nuclei were stained with DAPI. Single and merged colors recorded at a Pathway 855 (BD) are shown. Note the complete loss of nuclear CREB in dividing cells (white arrow heads). The bar represents 100 μm; Magnification: 20x. Morphology of the cells cultivated under normoxia or hypoxia (24 h each) was recorded by microscopic pictures. The pictures were taken on living, non-stained cells (transmitted). C. 1 mg isolated intact mitochondria were incubated in 1% Triton X100 and/or 10 U proteinase K in proteinase K buffer and were incubated at 37°C for 30 min and 60°C for 10 min. The reaction was stopped by boiling the probes in Laemmli buffer for 5 min and the stability of CREB was analysed by Western blot using an anti-CREB-specific mAb as described in Materials and Methods. D. Mitochondria were sub fractionated into outer membrane (OM), inter membrane space (IMS), inner membrane (IM) and mitochondrial matrix (MM). The purity of the fractions was analysed with marker proteins: TOM20 for OM, AIF for IMS, COXIV for IM, PDK4 for MM. E. Cells were cultured in the presence or absence of 50 ng/ml Leptomycin B, 10 μM LY294002 and 10 μM PYR-41 for 24 h under hypoxic conditions. Then cellular proteins were fractionated as described in the Material and Methods section. Additionally to the marker proteins used in (A) AIF served as a marker for the mitochondrial fraction. “W” represents the whole cell lysate, “C” the cytosolic fraction, “M” the membrane/mitochondrial fraction and “N” the nuclear fraction. The picture shows one of two experiments. An accumulation of highly modified CREB and the nuclear translocation of MEK1 and partially of AIF was found under leptomycin B treatment.

Article Snippet: TOM20 , Rabbit , Biorbyt (#orb128858) , polyclonal , Western Blot , 1:1000 , 5 % (w/v) BSA in TBS-T.

Techniques: Western Blot, Membrane, Incubation, Staining, Isolation, Marker, Cell Culture, Modification, Translocation Assay

Journal: Oncotarget

Article Title: Hypoxia-mediated alterations and their role in the HER-2/neuregulated CREB status and localization

doi: 10.18632/oncotarget.10474

Figure Lengend Snippet: Primary and labeled secondary antibodies used in this study

Article Snippet: TOM20 , Rabbit , Biorbyt (#orb128858) , polyclonal , Western Blot , 1:1000 , 5 % (w/v) BSA in TBS-T.

Techniques: Labeling, Western Blot, Staining, Flow Cytometry, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Exercise improved P2Y12-regulated microglial dynamics during stroke via endocannabinoid signaling

doi: 10.1101/2023.04.24.538065

Figure Lengend Snippet: Microglia interacted with neurons, inhibited neuronal calcium hyperactivity, and promoted neuronal mitochondria. A. Left panel: Representative images showing GCaMP6s signal changes in neurons imaged at 24 h after transient middle cerebral artery occlusion. Right panel: Representative images about the relative immunofluorescent intensity changes of GCaMP6s-dTomato. B. Comparisons of the amplitudes and frequencies of GCaMP6s signal changes in the vehicle, PE, and PE+PSB0739 groups. C. Representative images of the neuronal mitochondria and the microglia-neuron membrane junction captured using transmission electron microscope (TEM). Upper panel: images captured using 1200X objective, red box labeled the magnified site showed in the below panel. below panel: images captured using 11000X objective, red arrows indicates the neuronal mitochondria, purple box in PE group indicates the microglia-neuron junction. D. Comparisons of the relative expression of TOM20 among the vehicle, PE, and PE+PSB0739 groups. E. Comparisons of the relative expression of iNOS among the vehicle, PE, and PE+PSB0739 groups. F. Comparisons of the relative expression of TNF α and IL-10 among the vehicle, PE, and PE+PSB0739 groups. Each dataset is expressed as the mean ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. n = 6 mice.

Article Snippet: The sections were then incubated overnight with primary antibody [1:100 Mouse Anti-Neun, MAB377, Millipore, Massachusetts, USA; 1:100 Chicken Anti-Neun, ab134014, Cambridge, England; 1:400 Rbbit anti-ionized calcium binding adapter molecule 1 (Iba1), Wako, Tokyo, Japan; 1:100 Mouse anti-P2Y12, ab233760, Cambridge, England; 1:100 Rabbit anti-TOMM20, ab186735, Cambridge, England; 1:100 Rabbit anti-Cannabinoid Receptor I, ab259323, Cambridge, England; 1:100 Mouse anti-cannabinoid receptor 2, H00001269-M01, Novus, Colorado, USA; 1:100 Rabit Anti-Synaptophysin, ab32127, Cambridge, England], and then with a secondary antibody at room temperature for 1 h. Apoptosis was detected using a TUNEL Kit (TUNEL Apoptosis Detection Kit; KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s protocol.

Techniques: Membrane, Transmission Assay, Microscopy, Labeling, Expressing

Journal: bioRxiv

Article Title: Exercise improved P2Y12-regulated microglial dynamics during stroke via endocannabinoid signaling

doi: 10.1101/2023.04.24.538065

Figure Lengend Snippet: URB597 mimicked the improvement of microglial-neuron interactions induced by physical exercise, while AM630 decreased the protection of PE. A. Representative images showing GCaMP6s signal changes in neurons imaged at 24 h after transient middle cerebral artery occlusion.. B. Representative images about the immunofluorescent intensity changes of GCaMP6s-dTomato. C. Comparisons of the amplitudes and frequencies of the GCaMP6s signal changes among the vehicle, PE, URB597 and PE+AM630 groups. D. Representative images of the neuronal mitochondria and the microglia-neuron membrane junction captured by transmission electron microscope (TEM). Upper panel: images captured by1200X objective, red box labeled the magnified site showed in the below panel. below panel: images captured using 11000X objective, red arrows indicates the neuronal mitochondria and purple box in PE and URB597 group labeled the microglia-neuron junction. E. Comparison of TOM20 expression among the vehicle, PE, URB597 and PE+AM630 groups (Each dataset is expressed as the mean ± SD. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. n = 6 mice)

Article Snippet: The sections were then incubated overnight with primary antibody [1:100 Mouse Anti-Neun, MAB377, Millipore, Massachusetts, USA; 1:100 Chicken Anti-Neun, ab134014, Cambridge, England; 1:400 Rbbit anti-ionized calcium binding adapter molecule 1 (Iba1), Wako, Tokyo, Japan; 1:100 Mouse anti-P2Y12, ab233760, Cambridge, England; 1:100 Rabbit anti-TOMM20, ab186735, Cambridge, England; 1:100 Rabbit anti-Cannabinoid Receptor I, ab259323, Cambridge, England; 1:100 Mouse anti-cannabinoid receptor 2, H00001269-M01, Novus, Colorado, USA; 1:100 Rabit Anti-Synaptophysin, ab32127, Cambridge, England], and then with a secondary antibody at room temperature for 1 h. Apoptosis was detected using a TUNEL Kit (TUNEL Apoptosis Detection Kit; KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s protocol.

Techniques: Membrane, Transmission Assay, Microscopy, Labeling, Comparison, Expressing

Journal: Human Cell

Article Title: Astragaloside IV alleviates senescence of vascular smooth muscle cells through activating Parkin-mediated mitophagy

doi: 10.1007/s13577-022-00758-6

Figure Lengend Snippet: AS-IV improved mitophagy and attenuated mitochondrial membrane potential loss in BLM-induced VSMCs. A After administration of AS-IV, MMP was identified by TMRM staining in BLM-induced VSMCs. Magnification, 200 × , scale bar = 50 μm. B TEM was used to observe the influence of AS-IV on the mitochondrial structure of BLM-induced VSMCs. C Flow Cytometer was used to assess the impact of AS-IV on the TMRM + fluorescent intensity in BLM-stimulated VSMCs. D P62 expression was examined by Western blot in BLM-induced VSMCs, which were given AS-IV, and the P62/TOMM20 value was determined. E expression of Parkin was measured by Western blot in BLM-induced VSMCs. LD low dose, MD medium dose, HD high dose. * P < 0.05, ** P < 0.05, *** P < 0.001

Article Snippet: The primary antibodies included p16 (Beyotime, AF1069, 1:1000), p21 (ProteinTech, 10,355–1-AP, 1:3000), DcR2 (Boster, A05136, 1:1500), P62 (ProteinTech, 18,420–1-AP, 1:3000), TOMM20 (Boster, BM4366, 1:2000), Drp1 (Abcam, ab184247, 1:1000), and Parkin (Boster, PB9307, 1:1500).

Techniques: Membrane, Staining, Flow Cytometry, Expressing, Western Blot