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