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: Cell Proliferation

Article Title: Autophagy regulates cisplatin‐induced stemness and chemoresistance via the upregulation of CD 44, ABCB 1 and ADAM 17 in oral squamous cell carcinoma

doi: 10.1111/cpr.12411

Figure Lengend Snippet: Mitophagy regulates chemoresistance in FaDu cells. Immunofluorescence expression (A) and quantification of outer mitochondrial marker protein TOM20 in FaDU‐P and FaDu‐CDDP‐R (B) were analysed. Western blot analysis of inner mitochondrial marker protein COX‐IV was investigated (C). The mitochondrial delivery to lysosome in order to measure the mitophagic flux (in terms of increased red puncta) was assayed by employing mKeima‐Red‐Mito7 (D and E). Immunofluorescence expression of TOM20 with quantification of % of cells with less/no mitochondrial TOM20 staining in FaDU‐P and FaDu‐CDDP‐R cells after transient transfection of siControl and siATG14 are depicted (F and G). Western blot analysis of COX‐IV expression in ATG14‐deficient FaDU‐P and FaDu‐CDDP‐R cells is represented (H). Densitometry was performed on the original blots, the ratio of protein to actin in control cells was considered as 1. **P value < 0.01 was considered significant when compared between FaDu‐P and FaDu‐CDDP‐R groups. ## P value < 0.01 was considered significant when compared between FaDu‐CDDP‐R and siATG14‐ FaDu‐CDDP‐R

Article Snippet: Immunocytofluorescence staining and analysis FaDu‐P and FaDu‐CDDP‐R cells were fixed with 10% formaldehyde, permeabilized by 0.1% Triton X‐100, and then incubated with the primary antibodies for CD44 (1:500, Immunotools # 21810441), ABCB1 (1:500, abcam # ab155421), ADAM17 (1:500, abcam # ab574821), β‐catenin (1:500, BD Biosciences # 610153), LC3B (1:500; Novus Biologicals # NB‐100‐2220), TOM20 (1:500, BD Biosciences # 612278).

Techniques: Immunofluorescence, Expressing, Marker, Western Blot, Staining, Transfection

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