Journal: Cancer Science

Article Title: Gastric cancer–secreted exosomal X26nt increases angiogenesis and vascular permeability by targeting VE‐cadherin

doi: 10.1111/cas.14740

Figure Lengend Snippet: Exosomes derived from gastric cancer (GC) cells can be internalized by human umbilical vein endothelial cells (HUVECs). A, Representative transmission electron microscopy image of exosomes derived from BGC‐823 cells (BGCs) (scale bar, 100 nm). B, Particle diameter (nm) of the purified exosomes. C, Western blot of exosome markers of BGC cellular protein and corresponding exosomes. D, Exosomes from BGCs can fuse with HUVECs. DAPI and PKH67 were used to stain HUVECs and exosomes, respectively, and PKH67‐labelled exosomes were shown to enter HUVECs at 12 h. Arrows refer to PKH67‐labelled exosomes (scale bar, 50 μm; n = 3)

Article Snippet: HUVECs were obtained from ScienCell Research Laboratories and cultured in basal endothelial cell medium (EBM2) supplemented with the EGM‐2‐MV bullet kit (CC‐3156; Lonza).

Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Purification, Western Blot, Staining

Journal: Cancer Science

Article Title: Gastric cancer–secreted exosomal X26nt increases angiogenesis and vascular permeability by targeting VE‐cadherin

doi: 10.1111/cas.14740

Figure Lengend Snippet: The role of exosomes in angiogenesis. Human umbilical vein endothelial cells (HUVECs) were incubated with exosomes isolated from BGC‐823 cells (BGCs), and the proliferation, migration, and tube formation were examined after 24 h. To downregulate the level of exosomal (X26nt), we isolated exosomes from BGCs transfected with X26nt inhibitors (X26nt asRNA) for 24 h (exo X26nt del), and the scrambled X26nt inhibitors were transfected as control (exo I.NC). A, Proliferation of HUVECs measured by EdU assay (n = 3). B, Quantitative analysis EdU/DAPI rate of (A) (n = 3), * P < .05; ** P < .01. C, Migration of HUVECs measured by wound healing assay at 0 and 12 h (n = 3). D, Quantitative analysis wound closure rate of (C). Wound closure rate was defined as the percentage of the area occupied by the migrated cells to that of the empty area created by the scratching (n = 3), ** P < .01. E, Migration of HUVECs measured by transwell assay (n = 3). F, Quantitative analysis of migrated cells per area of (E) (n = 3), ** P < .01. G, Tube formation of HUVECs (n = 3). H, Quantitative analysis of branches per area of (G) (n = 3), ** P < .01

Article Snippet: HUVECs were obtained from ScienCell Research Laboratories and cultured in basal endothelial cell medium (EBM2) supplemented with the EGM‐2‐MV bullet kit (CC‐3156; Lonza).

Techniques: Incubation, Isolation, Migration, Transfection, Control, EdU Assay, Wound Healing Assay, Transwell Assay

Journal: Cancer Science

Article Title: Gastric cancer–secreted exosomal X26nt increases angiogenesis and vascular permeability by targeting VE‐cadherin

doi: 10.1111/cas.14740

Figure Lengend Snippet: The role of X26nt in angiogenesis. Human umbilical vein endothelial cells (HUVECs) were transfected directly with X26nt mimics (M.X26nt) or inhibitors (X26nt asRNA), and the corresponding scrambled mimics and inhibitors were used as controls (NC). Subsequently, the proliferation, migration, and tube formation of HUVECs were examined at 24 h. A, Proliferation of HUVECs measured by EdU assay (n = 3). B, Quantitative analysis of (A) (n = 3), * P < .05; ** P < .01. C, Migration of HUVECs measured by wound healing assay at 0 and 12 h (n = 3). D, Quantitative analysis of (C) (n = 3), ** P < .01. E, Migration of HUVECs measured by transwell assay (n = 3). F, Quantitative analysis of (E) (n = 3), * P < .05; ** P < .01. G, Tube formation of HUVECs (n = 3). H, Quantitative analysis of (G) (n = 3), ** P < .01

Article Snippet: HUVECs were obtained from ScienCell Research Laboratories and cultured in basal endothelial cell medium (EBM2) supplemented with the EGM‐2‐MV bullet kit (CC‐3156; Lonza).

Techniques: Transfection, Migration, EdU Assay, Wound Healing Assay, Transwell Assay

Journal: Cancer Science

Article Title: Gastric cancer–secreted exosomal X26nt increases angiogenesis and vascular permeability by targeting VE‐cadherin

doi: 10.1111/cas.14740

Figure Lengend Snippet: X26nt derived from exosomes decreases the expression of vascular endothelial cadherin (VE‐cadherin). A, Relative levels of X26nt in GES‐1 cell exosomes, MKN‐45 exosomes, MGC‐803 exosomes, and BGC‐823 exosomes by qRT‐PCR (n = 3), * P < .05; ** P < .01. B, The level of X26nt in human umbilical vein endothelial cells (HUVECs) cocultured with different exosomes were determined by qRT‐PCR (n = 3), ** P < .01. C, Exosomes derived from BGC‐823 cells (BGCs) transfected with Cy3‐labelled X26nt or without transfection (control) were added to HUVECs culture medium. HUVECs were fixed and stained with DAPI and Alexa 488 phalloidin. The fluorescence signal in HUVECs was detected by SP5 confocal microscope. Arrows refer to Cy3‐labelled X26nt exosomes (n = 3). D, Immunoblots of VE‐cadherin in HUVECs cocultured with different exosomes. GAPDH was presented as the loading control. Densitometric analysis of VE‐cadherin/GAPDH (n = 3) is shown, ** P < .01. E, Immunofluorescence of VE‐cadherin in HUVECs cocultured with different exosomes (n = 3). F, Quantitative analysis of the FITC‐dextran passage of HUVECs monolayer cocultured with different exosomes (n = 6), * P < .05. G, Green fluorescent protein‐mouse forestomach carcinoma (GFP‐MFC) cells were added to the top of HUVECs monolayer cocultured with different exosomes for 24 h that were grown on transwell inserts and incubated for another 12 h. The migratory GFP‐MFC cells were captured using a fluorescent microscope (n = 3). H, Quantitative analysis of the migratory GFP‐MFC cells of (G) (n = 3), ** P < .01

Article Snippet: HUVECs were obtained from ScienCell Research Laboratories and cultured in basal endothelial cell medium (EBM2) supplemented with the EGM‐2‐MV bullet kit (CC‐3156; Lonza).

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Transfection, Control, Staining, Fluorescence, Microscopy, Western Blot, Immunofluorescence, Incubation

Journal: Cancer Science

Article Title: Gastric cancer–secreted exosomal X26nt increases angiogenesis and vascular permeability by targeting VE‐cadherin

doi: 10.1111/cas.14740

Figure Lengend Snippet: X26nt directly targets and inhibits vascular endothelial cadherin (VE‐cadherin) expression. A, Schematic showing potential X26nt binding sites in the VE‐cadherin mRNA 3′UTR. B, The luciferase assay of HEK293T cells cotransfected firefly luciferase reporter plasmid containing either wild‐type (WT) or mutant VE‐cadherin mRNA 3′UTR with M.X26nt (n = 3), ** P < .01; n.s., nonsignificant. C, Western blot analysis of VE‐cadherin expression in human umbilical vein endothelial cells (HUVECs) transfected with M.X26nt. GAPDH was presented as the loading control. Densitometric analysis of VE‐cadherin/GAPDH (n = 3) is shown, * P < .05. D, Western blot analysis of VE‐cadherin expression in HUVECs transfected with X26nt asRNA. GAPDH was presented as the loading control. Densitometric analysis of VE‐cadherin/GAPDH (n = 3) is shown, * P < .05. E, Immunofluorescence staining of VE‐cadherin in HUVECs transfected with M.X26nt and X26nt asRNA (n = 3)

Article Snippet: HUVECs were obtained from ScienCell Research Laboratories and cultured in basal endothelial cell medium (EBM2) supplemented with the EGM‐2‐MV bullet kit (CC‐3156; Lonza).

Techniques: Expressing, Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Western Blot, Transfection, Control, Immunofluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: MMP14-Containing Exosomes Cleave VEGFR1 and Promote VEGFA-Induced Migration and Proliferation of Vascular Endothelial Cells

doi: 10.1167/iovs.18-26277

Figure Lengend Snippet: Migration and VEGFA-induced proliferation of HUVECs with exposure to WT-derived exosomes and Δexon4-derived exosomes. (A) HUVEC migration toward medium containing different amounts of WT-derived exosomes or Δexon4-derived exosomes. Medium only was used as a negative control, and complete medium including supplements was used as a positive control. (B) Protein content of WT-derived exosomes vs. Δexon4-derived exosomes. (C) HUVEC migration toward medium containing WT-derived exosomes or Δexon4-derived exosomes in amounts corresponding to equal protein concentrations. (D) VEGFA-induced HUVEC proliferation rate after treatment with WT-derived exosomes or Δexon4-derived exosomes.

Article Snippet: Endothelial cell growth medium including supplements (EBM-2; Lonza) was used to maintain HUVEC (ScienCell).

Techniques: Migration, Derivative Assay, Negative Control, Positive Control