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: 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