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: Physiological Research

Article Title: Analysis of Gene Expression Profiles Regulating Phenotypic Transformation of Vascular Smooth Muscle Cells by Endothelial Cell-Derived Exosomes

doi: 10.33549/physiolres.935541

Figure Lengend Snippet: A ) TEM (×30,000, voltage = 100.0 kV) reveals that ECs exosomes have a double-concave disc structure. B ) NTA showed an average diameter of 85.45 nm, with 99.33 % of the exosomes at this size, consistent with typical extracellular vesicles (30–150 nm). C )WB analysis confirmed high expression of exosome marker proteins CD9, CD63, and CD81 in ECs exosomes.

Article Snippet: The complete medium for rat aortic ECs ( Procell Life Science & Technology Co., Ltd” (Wuhan, China, CM-R075)), it contained 5 % FBS, 50ng/mL VEGF, 10ng/mL EGF, 20ng/mL bFGF and 1 % penicillin and streptomycin,CD9, CD31, CD63, and CD81 monoclonal antibodies (Abcam, USA), fluorescein (Cy3)-labeled goat anti-rabbit IgG (Boster, Wuhan, China), exosome extraction kit (exoEasy Maxi Kit, Qiagen), GW4869 exosome inhibitor (Yensen, China), SW-CJ-IF clean bench, constant temperature CO 2 incubator (Thermo, USA), 6-well Transwell culture plate (0.4 mm) (Costar 3450, USA), Olympus inverted microscope, fluorescence microscope, Agilent 2100 Bioanalyzer (Agilent Technologies, USA), and NovaSeq 6000 (Illumina, USA) were employed in this investigation.

Techniques: Expressing, Marker