Journal: Viruses

Article Title: Cargo and Biological Properties of Extracellular Vesicles Released from Human Adenovirus Type 4-Infected Lung Epithelial Cells

doi: 10.3390/v17101300

Figure Lengend Snippet: Characterization of the EV populations isolated from the mock- and AdV-infected A549 cells. ( A ) Phase contrast images of the A549 cells showing the magnitude of cytopathic effect at 24 h post-infection. Cells were infected at MOI = 10. Scale bars = 100 µm. ( B ) The representative Western blot analysis showing enrichment of the EV markers CD63, CD9, CD81, Alix in EVs-Ctr and EVs-AdV, compared to their cells of origin (cell lysates). The ER marker GRP94 was used to assess the presence of non-EV contaminants. Images are representative of three different experiments. ( C ) The TEM images of EVs isolated by magnetic immunocapture form the supernatants of mock-infected cells (Ctr-EVs) or HAdV-E4-infected (AdV-EVs) A549 cells. The magnetic beads are recognizable by their opaque appearance and uniform size of 50 nm (black arrows). Scale bars = 200 nm. ( D ) The TEM image of the purified HAdV-E4 particles (white arrows). Scale bar = 200 nm. ( E ) The fluorescent nanoparticle tracking analysis of Ctr-EVs (left panel), AdV-EVs (middle panel), and magnetic beads alone (right panel).

Article Snippet: EVs were isolated from the concentrated medium with EV isolation kits Pan (human), or CD81 human (Cat # 130-111-572 and 130-110-914, Miltenyi Biotec, Auburn, CA, USA), according to the manufacturer’s instructions.

Techniques: Isolation, Infection, Western Blot, Marker, Magnetic Beads, Purification

Journal: Oncogene

Article Title: CYP4A in tumor-associated macrophages promotes pre-metastatic niche formation and metastasis

doi: 10.1038/onc.2017.118

Figure Lengend Snippet: M2 macrophage-derived factors regulated by CYP4A drive lung pre-metastatic niche formation. ( a ) TGF-β, IGF-1, VEGF, SDF-1, PDGF-B, MMP-2 and MMP-9 in M2 macrophages (M2) and CYP4A10 high M2 were determined by qPCR. ( b ) VEGF and SDF-1 from the culture supernatants were determined by ELISA. ( c ) The conditioned medium (CM) from M2 or CYP4A10 high M2 incubated with or without neutralizing anti-VEGF (20 μg/ml) or anti-SDF-1 (10 μg/ml) antibodies for 48 h was added to the lower chamber. VEGFR1 + myeloid cells were added to the upper chamber, and then incubated over night. The cells migrated to the lower chamber were counted. ( d ) L929 fibroblast (L929) and lung-derived fibroblast (LF) were stimulated with the CM from CYP4A10 high M2 with or without neutralizing anti-TGF-β (10 μg/ml), anti-VEGF (20 μg/ml), anti-SDF-1 (10 μg/ml), anti-PDGF-B (10 μg/ml) or anti-MMP-9 (20 μg/ml) antibodies for 48 h. α-SMA was determined by qPCR. ( e ) L929 and LF were pre-treated with or without TGF-β receptor inhibitor (A83-01, 10 μ M ) for 1 h, and then incubated with the CM from CYP4A10 high M2 for another 48 h. α-SMA and fibronectin were measured by western blot. ( f ) L929 and LF were stimulated with the CM from M2 or CYP4A10 high M2 with or without a combination of neutralizing anti-TGF-β (10 μg/ml), anti-VEGF (20 μg/ml) and anti-SDF-1 (10 μg/ml) antibodies for 48 h. α-SMA was determined by qPCR. ( g ) M2 CM, CYP4A10 high M2 CM, M2 CM plus VEGF (800 pg/ml) or SDF-1 (60 pg/ml) or TGF-β (200 pg/ml), and M2 CM plus combination cytokines (VEGF, SDF-1 and TGF-β) were intraperitoneally injected into BALB/c mice daily for 2 weeks. VEGFR1 + myeloid cells in the lungs were analyzed by flow cytometry ( n =5). The value is presented as the mean±s.e.m. # P <0.05, ## P <0.01 vs M2, ^ P <0.05, ^^ P <0.01 vs CYP4A10.

Article Snippet: Mouse recombinant VEGF, SDF-1 and TGF-β were purchased from R&D Systems (Oxford, UK).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Injection, Flow Cytometry

Journal: Oncogene

Article Title: CYP4A in tumor-associated macrophages promotes pre-metastatic niche formation and metastasis

doi: 10.1038/onc.2017.118

Figure Lengend Snippet: CYP4A/20-HETE–STAT3 signaling is crucial for M2 polarization of macrophages and migration of VEGFR1 + myeloid cells in vitro . ( a , b ) Mouse peritoneal macrophages (PNMS) or human monocyte-derived macrophages (MDMs) were stimulated with IL-4/IL-13 (20 ng/ml) for 12 h or the tumor-conditioned medium (CM) for 72 h to generate M2 macrophages, and then treated with HET0016 (5 μ M ), DDMS (10 μ M ) or vehicle for 24 h. STAT3 and p-STAT3 was determined by western blot. ( c ) Immunostaining analysis of CD206 (red) and p-STAT3 (green) in PNMS from the above treated groups was shown. Scale bars, 20 μm. ( d ) The effect of siRNA against STAT3 on the protein levels of STAT3 was assayed by western blot. ( e ) The effect of Stattic on p-STAT3 was assayed by western blot. ( f ) MDMs and PNMS were pre-treated with or without STAT3 inhibitor (Stattic, 20 μ M ) for 1 h, and then incubated with IL-4/IL-13 for 12 h or the tumor CM for 72 h, followed by treatment with or without 20-HETE (1 μ M ) for another 24 h. In another experiment, MDMs and PNMS were pre-treated with or without STAT3 siRNA (STAT3-siRNA) and scramble siRNA for 24 h, and then incubated with IL-4/IL-13 for 12 h or the tumor CM for 72 h, followed by treatment with 20-HETE (1 μ M ) for another 24 h. Th2 cytokines (TGF-β and IL-10) were determined by ELISA. ( g ) VEGF and SDF-1 from the culture supernatants in the above groups were determined by ELISA. ( h ) The CM in the above treated groups was added to the lower chamber. VEGFR1 + myeloid cells were added to the upper chamber, and then incubated over night. The cells migrated to the lower chamber were counted. Each value presents the mean±s.e.m. of three independent triplicate experiments. * P <0.05, ** P <0.01 vs control, # P <0.05, ## P <0.01 vs CYP4A, ^ P <0.05, ^^ P <0.01 vs 20-HETE.

Article Snippet: Mouse recombinant VEGF, SDF-1 and TGF-β were purchased from R&D Systems (Oxford, UK).

Techniques: Migration, In Vitro, Derivative Assay, Western Blot, Immunostaining, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: Oncogene

Article Title: CYP4A in tumor-associated macrophages promotes pre-metastatic niche formation and metastasis

doi: 10.1038/onc.2017.118

Figure Lengend Snippet: A proposed mechanism to explain the role of CYP4A/20-HETE in TAMs in pre-metastatic niche formation and metastasis. 20-HETE, 20-hydroxyeicosatetraenoic acid; CYP4A, cytochrome P450 4A; MMP-9. matrix metalloproteinase-9; SDF-1, stromal cell-derived factor-1; STAT3, signal transducer and activator of transcription 3; TAMs, tumor-associated macrophages; TGF-β, transforming growth factor-β VEGF, vascular endothelial growth factor.

Article Snippet: Mouse recombinant VEGF, SDF-1 and TGF-β were purchased from R&D Systems (Oxford, UK).

Techniques: Derivative Assay