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

Journal: Journal of Nanobiotechnology

Article Title: Cardiac-derived extracellular vesicles improve mitochondrial function to protect the heart against ischemia/reperfusion injury by delivering ATP5a1

doi: 10.1186/s12951-024-02618-x

Figure Lengend Snippet: cEVs alleviated ferroptosis and mitochondrial dysfunction in cardiomyocytes under oxidative stress in vivo. A Prussian blue staining showing decreased iron accumulation in mice heart treated with cEVs compared to treated with PBS at day 3 after MI/R (scale bar = 50 μm, n = 6). B Evaluation of 4-HNE accumulation in MI/R mice heart of different groups detected by IHC staining (scale bar = 50 μm, n = 6). C MitoSOX staining indicating mitochondria superoxide production in mice heart ( n = 4). D Relative mRNA expression of Ptgs2 in mice heart of different groups detected by RT-qPCR ( n = 5). E, F Relative protein expression of GPX4 in mice heart of different groups detected by western blot and corresponding quantification analysis ( n = 4). G Representative TEM images showing the mitochondrial morphology in MI/R mice heart (scale bar = 1 μm, n = 5). H, I Mitochondrial area ( H ) and mitochondrial length/width ratio ( I ) detected in TEM images. J, K Relative mRNA expression of TFAM ( J ) and PGC-1α ( K ) in mice heart of different groups detected by RT-qPCR ( n = 5). L Representative micrographs of Tom20 IHC staining in mice heart at day 3 after operation (scale bar = 100 μm, n = 6). (Data were expressed as Mean ± SD. Statistically significant: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Consequently, the PVDF membrane was blocked with 5% nonfat milk for 1 h at room temperature, and then incubated with a primary antibody CD9 (Abcam, UK), Alix (Abcam, UK), TSG101 (Abcam, UK), Calnexin (Abcam, UK), ATP5a1 (Abclonal, China), GPX4 (Abclonal, China), Tom20 (Abclonal, China) and GAPDH (Santa Cruz Biotechnology, USA).

Techniques: In Vivo, Staining, Immunohistochemistry, Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Nanobiotechnology

Article Title: Cardiac-derived extracellular vesicles improve mitochondrial function to protect the heart against ischemia/reperfusion injury by delivering ATP5a1

doi: 10.1186/s12951-024-02618-x

Figure Lengend Snippet: cEVs attenuated oxidative stress-induced cell ferroptosis and mitochondrial dysfunction in vitro. A Schematic diagram showing MCM cells under the H/R model and cEVs treatment. B Representative micrographs showing cEVs uptaken by MCM cells 6 h after coculture. DiL was used to label cEVs, phallodin was used to label microfilament, DAPI was used to label nuclei (scale bar = 100 μm). C Representative micrographs of IF staining showing decreased lipid peroxidation in MCM cells after cEVs treatment (scale bar = 50 μm). D MDA production of MCM cells in different groups ( n = 5). E Relative Ptgs2 mRNA expression in MCM cells detected using RT-qPCR ( n = 3). F, G Relative GPX4 protein expression in MCM cells detected using western blot and corresponding quantification analysis ( n = 3). H JC-1 staining measuring the mitochondrial membrane potential of MCM cells. There were increased hyperpolarization (red) and decreased depolarization (green) of mitochondria in MCM cells following cEVs treatment (scale bar = 50 μm). I, J Relative Tom20 protein expression in MCM cells detected by western blot and corresponding quantification analysis ( n = 4). K-M Representative TEM images showing the mitochondrial morphology ( K ), mitochondrial area ( L ), and mitochondrial length/width ratio ( M ) in MCM cells detected in TEM images (scale bar = 1 μm, n = 5). N, O Flow cytometry showing cEVs treatment reduced Mitochondrial ROS content in MCM cells ( n = 3). P Representative micrographs of MitoSOX IF staining showing decreased Mitochondrial ROS content in MCM cells after cEVs treatment (scale bar = 50 μm). (Data were expressed as Mean ± SD. Statistically significant: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Consequently, the PVDF membrane was blocked with 5% nonfat milk for 1 h at room temperature, and then incubated with a primary antibody CD9 (Abcam, UK), Alix (Abcam, UK), TSG101 (Abcam, UK), Calnexin (Abcam, UK), ATP5a1 (Abclonal, China), GPX4 (Abclonal, China), Tom20 (Abclonal, China) and GAPDH (Santa Cruz Biotechnology, USA).

Techniques: In Vitro, Staining, Expressing, Quantitative RT-PCR, Western Blot, Membrane, Flow Cytometry

Journal: Journal of Nanobiotechnology

Article Title: Cardiac-derived extracellular vesicles improve mitochondrial function to protect the heart against ischemia/reperfusion injury by delivering ATP5a1

doi: 10.1186/s12951-024-02618-x

Figure Lengend Snippet: ATP5a1 was responsible for cEVs-mediated resistance to oxidative stress damage in cardiomyocytes. A Top 10 of enriched KEGG pathways of top 100 genes from RNA-seq sequencing data of cEVs samples ( n = 4). B Top 5 of GO BP, GO MF, and GO CC analysis of top 100 genes from RNA-seq sequencing data of cEVs samples ( n = 4). C KEGG chord diagram showing genes corresponding to different enrichment KEGG pathways. On the left is the genes, on the right is the mmu term information of the significant enrichment KEGG pathways. D Validation of RNA-seq sequencing results of cEVs samples detected by RT-qPCR ( n = 3). E Relative mRNA expression level of ATP5a1 in MCM cells following siRNA-1, siRNA-2, and siRNA-3 transfection ( n = 3). F Relative mRNA expression level of ATP5a1 in MCM cells after transfection of 50nM or 100nM siRNA-1, respectively ( n = 3). G Relative mRNA expression level of Ptgs2 in MCM cells of different groups ( n = 3). H MDA production in MCM cells ( n = 3). I, J Relative protein expression of ATP5a1, GPX4, and Tom20 in different groups detected by western blot and corresponding quantitative analysis ( n = 3). K Representative TEM images showing the mitochondrial morphology of MCM cells in different groups. L, M Relative mitochondrial length/width ratio ( L ) and mitochondrial area ( M ) detected in TEM images (scale bar = 1 μm, n = 6). N Representative micrographs of MitoSOX IF staining showing Mitochondrial ROS content in MCM cells after different treatment (scale bar = 50 μm). O, P Flow cytometry showing Mitochondrial ROS content in MCM Cells by MitoSOX and corresponding quantification analysis ( n = 3). Q JC-1 staining showing the mitochondrial membrane potential of MCM cells in different groups ( n = 5). R Mitochondrial oxidative respiration of MCM cells in different groups detected by the cellular OCR. S - V Basal Respiration ( S ), Maximal Respiration ( T ), ATP production ( U ), and Spare Respiratory Capacity ( V ) of MCM cells in different groups. (Data were expressed as Mean ± SD. Statistically significant: ns: not statistically significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Consequently, the PVDF membrane was blocked with 5% nonfat milk for 1 h at room temperature, and then incubated with a primary antibody CD9 (Abcam, UK), Alix (Abcam, UK), TSG101 (Abcam, UK), Calnexin (Abcam, UK), ATP5a1 (Abclonal, China), GPX4 (Abclonal, China), Tom20 (Abclonal, China) and GAPDH (Santa Cruz Biotechnology, USA).

Techniques: RNA Sequencing Assay, Sequencing, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Staining, Flow Cytometry, Membrane

Journal: Journal of Nanobiotechnology

Article Title: Cardiac-derived extracellular vesicles improve mitochondrial function to protect the heart against ischemia/reperfusion injury by delivering ATP5a1

doi: 10.1186/s12951-024-02618-x

Figure Lengend Snippet: cEVs alleviated H/R induced ferroptosis and mitochondrial damage in NMCMs by transporting ATP5a1. A Representative micrograph showing cEVs uptaken by isolated NMCMs. B Relative protein expression of ATP5a1, GPX4, and Tom20 of NMCMs in different groups detected by western blot ( n = 3). C-E Quantitative analysis of protein expression of ( C ) ATP5a1, ( D ) GPX4, and ( E ) Tom20 of NMCMs in different groups ( n = 3). F Mitochondrial oxidative respiration of NMCMs in different groups detected by the cellular OCR ( n = 6). G-J Basal Respiration ( G ), Maximal Respiration ( H ), ATP production ( I ), and Spare Respiratory Capacity ( J ) of NMCMs in different groups. K Representative micrographs of MitoSOX IF staining showing Mitochondrial ROS content in NMCMs (scale bar = 50 μm). L, M Flow cytometry detection showing Mitochondrial ROS content in NMCMs revealed by MitoSOX staining and corresponding quantification analysis ( n = 3). (Data were expressed as Mean ± SD. Statistically significant: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Consequently, the PVDF membrane was blocked with 5% nonfat milk for 1 h at room temperature, and then incubated with a primary antibody CD9 (Abcam, UK), Alix (Abcam, UK), TSG101 (Abcam, UK), Calnexin (Abcam, UK), ATP5a1 (Abclonal, China), GPX4 (Abclonal, China), Tom20 (Abclonal, China) and GAPDH (Santa Cruz Biotechnology, USA).

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

Journal: Journal of Nanobiotechnology

Article Title: Cardiac-derived extracellular vesicles improve mitochondrial function to protect the heart against ischemia/reperfusion injury by delivering ATP5a1

doi: 10.1186/s12951-024-02618-x

Figure Lengend Snippet: Overexpression of ATP5a1in ADSC improved the therapeutic efficacy of ADSC-EVs in murine MI/R models. A Representative image of cardiac function of mice treated with PBS, ADSC-EVs, or ADSC-EVs ATP5a1 determined by M-mode echocardiography at day 3 post MI/R operation ( n = 6–7). B-E Results of LVEF ( B ), LVFS ( C ), LVEDV ( D ), and LVESV ( E ) in mice detected by echocardiogram ( n = 6–7). F, G Representative TTC staining and corresponding quantification analysis showing infarcted area/AAR ratio of mice heart in different groups (scale bar = 1 mm, n = 5). H Representative H&E staining of mice heart in different groups (scale bar = 100 μm, n = 6). I Representative micrographs of Prussian blue staining showing decreased iron accumulation in mice heart after ADSC-EVs ATP5a1 treatment (scale bar = 50 μm, n = 6). J Representative micrographs of Tom20 IHC staining showing alleviated mitochondrial damage in mice heart after ADSC-EVs ATP5a1 treatment (scale bar = 100 μm, n = 6). (Data were expressed as Mean ± SD. Statistically significant: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Consequently, the PVDF membrane was blocked with 5% nonfat milk for 1 h at room temperature, and then incubated with a primary antibody CD9 (Abcam, UK), Alix (Abcam, UK), TSG101 (Abcam, UK), Calnexin (Abcam, UK), ATP5a1 (Abclonal, China), GPX4 (Abclonal, China), Tom20 (Abclonal, China) and GAPDH (Santa Cruz Biotechnology, USA).

Techniques: Over Expression, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: Functional characterization of the Met50Val substitution in SLC30A9 as a novel case of adaptive introgression in humans

doi: 10.1101/2022.06.29.498106

Figure Lengend Snippet: (A) Superresolution STED microscopy in cells transfected with ZnT9-50Met and ZnT9-50Val immunoassayed with anti-HA (green) and with the mitochondrial marker anti-TOM20. Scale bar = 10 µm. (B-C) Bar graph representing mean inter-mitochondrial distance (B) and relative mitochondria area (C) in cells transfected with ZnT9-50Met or ZnT9-50Val (n=10). *** p<0.001 using Bonferroni test between conditions. (D) Bar graph measuring FRET between the endoplasmic reticulum and mitochondria transfected with an empty vector (control), ZnT9-50Met or ZnT9-50Val together with the FEMP probe (n=46-65).

Article Snippet: We used the rabbit anti-human TOM20 and the rabbit anti-human Calreticulin antibodies (1:1000) and secondary antibodies Abberior STAR RED or ORANGE (1:350).

Techniques: Microscopy, Transfection, Marker, Plasmid Preparation

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