Journal: Scientific Reports

Article Title: Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

doi: 10.1038/srep10300

Figure Lengend Snippet: ( a ) Confocal microscopic observation of A431 cells treated with CD63-GFP-exosomes (20 μg/ml) in the presence or absence of EGF (500 nM) for 24 h at 37 °C. Green signals, CD63-GFP-exosomes; blue signals, Hoechst 33342 for nuclear staining. Scale bar, 20 μm. ( b ) Relative cellular uptake of CD63-GFP-exosomes in the same experimental condition of ( a ) analysed using a flow cytometer. ( c ) Internalisation of CD63-exosomes (20 μg/ml) and Texas Red-dextran (70 kDa, macropinocytosis marker) in the presence of EGF (500 nM) by A431 cells analysed using a confocal microscopy after treatment for 24 h at 37 °C. Arrows show representative colocalisation of exosomes and dextran inside cells. Scale bar, 10 μm. ( d ) Effects of the macropinocytosis inhibitor, EIPA (100 nM), on the cellular uptake of CD63-GFP-exosomes (20 μg/ml) with EGF (100 nM) for 3 h at 37 °C, analysed using a flow cytometer. ( b,d ) The data are the averages (±SD) of three experiments. ** p < 0.01, *** p < 0.001.

Article Snippet: The boiled samples were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Pittsburgh, PA, USA), and treated with anti-CD63 antibody (TS63, Abcam, Cambridge, UK).

Techniques: Staining, Flow Cytometry, Marker, Confocal Microscopy

Journal: Scientific Reports

Article Title: Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

doi: 10.1038/srep10300

Figure Lengend Snippet: ( a ) Relative cellular uptake of CD63-GFP-exosomes (20 μg/ml) in MIA PaCa-2 or BxPC-3 cells in the presence or absence of EGF (500 nM) for 24 h at 37 °C, analysed using a flow cytometer. ( b , c ) Relative cellular uptake of FITC-dextran ( b ) or FITC-transferrin ( c ) in MIA PaCa-2 or BxPC-3 cells in the absence of EGF for 24 h at 37 °C, analysed using a flow cytometer. ( a - c ) The data are the averages (±SD) of three experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. ( d ) Confocal microscopic observation of MIA PaCa-2 cells treated with CD63-GFP-exosomes (20 μg/ml) in the presence of EGF (500 nM) in same experimental condition of ( a ). Green signals, CD63-GFP-exosomes; red signals, Texas Red-labelled dextran; blue signals, Hoechst 33342 for nuclear staining. Scale bar, 10 μm.

Article Snippet: The boiled samples were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Pittsburgh, PA, USA), and treated with anti-CD63 antibody (TS63, Abcam, Cambridge, UK).

Techniques: Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

doi: 10.1038/srep10300

Figure Lengend Snippet: ( a ) Cell viability of A431 cells treated with CD63-GFP-exosomes (20 μg/ml) in serum-free cell culture medium with or without co-treatment of EGF (100 or 500 nM) for 24 h at 37 °C, analysed using a WST-1 assay. ( b ) Cytotoxicity of saporin-encapsulated exosomes (4 μg/ml) or saporin (7 μg/ml) with or without co-treatment of EGF (500 nM). A431 cells were treated with each test sample in 10% FBS containing cell culture medium for 48 h at 37 °C, prior to a WST-1 assay. The data are the averages (±SD) of three experiments. ** p < 0.01, *** p < 0.001.

Article Snippet: The boiled samples were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Pittsburgh, PA, USA), and treated with anti-CD63 antibody (TS63, Abcam, Cambridge, UK).

Techniques: Cell Culture, WST-1 Assay

Journal: Scientific Reports

Article Title: Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

doi: 10.1038/srep10300

Figure Lengend Snippet: ( a ) Confocal microscopic observation of A431 cells treated with EGF- or transferrin-encapsulated CD63-GFP-exosomes (20 μg/ml) for 24 h at 37 °C. Green signals, CD63-GFP-exosomes; blue signals, Hoechst 33342 for nuclear staining. Scale bar, 20 μm. ( b ) Relative cellular uptake of CD63-GFP-exosomes with encapsulation of EGF or transferrin in the exosomes in the same experimental condition with ( a ), prior to analysis using a flow cytometer. The data represent the average (±SD) of three experiments. *** p < 0.001.

Article Snippet: The boiled samples were separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Pittsburgh, PA, USA), and treated with anti-CD63 antibody (TS63, Abcam, Cambridge, UK).

Techniques: Staining, Encapsulation, Flow Cytometry

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A) A mammalian two-hybrid assay was performed by transfecting Vero cells with the indicated combinations of pACT and pBIND plasmids along with the pG5luc firefly luciferase reporter plasmid. At 48 h after transfection, the normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) was measured and represented as fold of the control activity, which was obtained by combination with the empty pACT or empty pBIND plasmid. Data are the mean ± SD of at least three independent experiments. (B) 293T cells were transfected for 24 h with FLAG-tagged IFITM1 and HA-tagged 3Cm, 2B, 2BC, 2C, 3A, or 3AB, or HA-tagged IFITM1 and FLAG-tagged TGN46 expression plasmids, as indicated, followed by coimmunoprecipitation (IP) with an anti-FLAG, -HA or control IgG antibody. The resulting immunoprecipitates and whole-cell lysates were subjected to immunoblotting (IB) with anti-FLAG and anti-HA antibodies. (C) Vero cells were transfected with FLAG-IFITM1. At 24 h after post-transfection, the cells were fixed and double stained with anti-IFITM and anti-EEA1, anti-CD63, or anti-LBPA antibodies, as indicated. Pearson correlation coefficient analyses for data were obtained from ≥10 cells. Correlation coefficients are presented as the mean and standard deviation. (D) Vero cells was transfected with FLAG-IFITM1 and HA-2B, HA-2BC, HA-2C, HA-3A, or HA-3AB. At 24 h after transfection, cells were fixed and stained with anti-FLAG and anti-HA antibodies. Bars, 4 μm. Pearson correlation coefficient analyses for data were obtained from 4–8 cells.

Article Snippet: Mouse monoclonal anti-CD63 antibody was from Novus Biologicals.

Techniques: Two Hybrid Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Control, Expressing, Western Blot, Staining, Standard Deviation

Journal: PLOS Pathogens

Article Title: IFITM1 enhances nonenveloped viral RNA replication by facilitating cholesterol transport to the Golgi

doi: 10.1371/journal.ppat.1011383

Figure Lengend Snippet: (A) A mammalian two-hybrid analysis was carried out to examine interactions between IFITM1 and ACBD3, PI4KB, OSBP, or CERT, and the results are shown as described in . Data are the mean ± SD of at least three independent experiments. (B) HA-tagged IFITM1 and FLAG-tagged ACBD3, PI4KB, OSBP, or CERT were cotransfected into 293T cells, and the cell lysates were subjected to immunoprecipitation with anti-HA antibody or control IgG. The resulting immunocomplexes and whole-cell lysates were detected by anti-FLAG and anti-HA antibodies. (C) Vero cells were transfected with FLAG-IFITM1. At 24 h, the cells were labeled with anti-FLAG and anti-ACBD3 (top), anti-PI4KB (middle), or anti-OSBP (bottom) antibodies. (D) Vero IFITM1 cells were incubated with Tet (−) or Tet (+) for 72 h, and the cells were fixed and double stained with the indicated antibodies. (E) Vero cells were transfected with HA-IFITM1. At 24 h, the cells were labeled using anti-HA, anti-OSBP and anti-EEA1, or anti-CD63 antibodies. Bars, 4 μm. Pearson correlation coefficient analyses for data were obtained from ≥10 cells. Correlation coefficients are presented as the mean and standard deviation (C-E).

Article Snippet: Mouse monoclonal anti-CD63 antibody was from Novus Biologicals.

Techniques: Immunoprecipitation, Control, Transfection, Labeling, Incubation, Staining, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes

doi: 10.1128/spectrum.02452-21

Figure Lengend Snippet: Immune recognition of various EV-D68 densities and characterization of membrane-associated virus. (A) The y axis represents average dilutions of anti-EV-D68 mouse serum required to neutralize virus, divided by the average TCID 50 for respective viral densities (ANOVA post hoc Student'’s t test, P = 0.42, P = 0.68, P = 0.70). (B) Three viral density isolates (1.11, 1.20, and 1.24 g/cm 3 ) were treated with 0.01 mg/mL 15C5-Chmra antibody for 1 h, then mix was put onto TCID 50 plates to assess the viral titer of each isolate. Gray highlight represents detection limit. Asterisks (*) indicate statistical significance (1.11 g/cm 3 , P = 0.0003; 1.20 g/cm 3 , P < 0.0001; 1.24 g/cm 3 , P = 0.0064), all versus respective control, determined by Dunnett’s Method. (C) 15C5-Chmra antibody bound to magnetic beads was added to membrane-associated and naked virus. After 1 h, a magnet was used to remove antibody and the supernatant was added to a TCID 50 plate to assess viral titer (15C5-Chmra versus control: *, P < 0.0005 for both membrane-associated and naked virus; Dunnett’s Method). (D) ICAM-5 or N -acetylneuraminic acid (sialic acid) were attached to magnetic beads and the antibody/bead complex was incubated with membrane-associated or naked virus samples for 1 h. Beads were rinsed twice in excess PBS and viral titer was assessed to determine how much virus was immunoprecipitated from the supernatant (control versus ICAM5 and control versus sialic acid for membrane-associated and naked virus; *, P = 0.0001 determined by Dunnett’s Method). (E) Exosome antibody array on 1.11 g/cm 3 fraction, examining cytosolic proteins (FLOT1, ALIX, TSG101), transmembrane proteins (CD63, CD81, ANXA5), and cis -golgi matrix protein as markers for cellular contamination (GM130). Example blot is shown on the right and chart represents average intensity across three biological replicates. Positive control indicates detection reagents are working correctly, and do not represent an exosome-specific control. Error bars represent standard deviation. Statistics: comparison with control (blank) using Dunnett’s Method ( P = 0.999 for GM130; *, P = 0.027 for FLOT1; P = 0.218 for ICAM; *, P = 0.005 for ALIX; P = 0.086 for CD81; *, P < 0.0001 for CD63; P = 0.305 for EpCAM; *, P < 0.0001 for ANXA5; *, P = 0.0008 for TSG101). Asterisks indicate statistical significance. (F) Anti-CD81 or anti-CD63 antibodies were attached to magnetic beads and incubated with membrane-associated virus. Supernatant was discarded, and beads were rinsed and treated with 0.01% NP-40 (to dissolve exosomes and release virus from bead) before TCID 50 measurement. CD81 versus control: *, P = 0.0178; CD63 versus control: *, P = 0.0180 as determined by Dunnett’s Method. Gray highlight represents detection limit. (G) RD or SH-SY5Y cells in TCID 50 plate were infected with MO47 with or without exosomes in the medium. The “A549 exosomes added” bar represents exosome-depleted media to which purified A549 exosomes were added. Gray panel represents TCID 50 plates containing SH-SY5Y cells. Each condition represents 3 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05. Green panel represents TCID 50 plates containing RD cells. Each condition represents 4 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05.

Article Snippet: We followed the coupling protocol from the Dynabeads Antibody Coupling Kit (Thermo Fisher, cat no. 14311D) to covalently attach magnetic beads to the following antibodies: anti-CD81 (1D6) monoclonal antibody (Novus Biologicals NB100-65805), anti-CD63 (H5C6) monoclonal antibody (Novus Biologicals NBP2-42225), 15C5-chimeric monoclonal antibody (generous gift from Michael Pauly at ZabBio), and anti-HSV negative control (generous gift from Michael Pauly at ZabBio).

Techniques: Membrane, Virus, Control, Magnetic Beads, Incubation, Immunoprecipitation, Ab Array, Positive Control, Standard Deviation, Comparison, Infection, Purification