Journal: Journal of Biomedical Science
Article Title: CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells
doi: 10.1186/s12929-024-01084-9
Figure Lengend Snippet: CD81-guided fusion proteins are cargo of secreted EV populations. A Representative immunoblotting of cell and EV lysates (1 μg proteins/well). EVs are positive to transmembrane (CD9) and cytosolic proteins (SYNTENIN, TSG101), while negative to CALNEXIN, and with low detectable levels of GAPDH compared to cell lysates. B Dot plots of imaging flow cytometry to detect GFP-positive EVs. The green fluorescent signal (Ch02, 488 nm laser) was detected as sub-gating of EVs labeled with Cell Mask Deep Red (CMDR, in orange, Ch11, 635 nm) to side-scatter (Ch06). Non-fluorescent, calibrator SpeedBeads, Amnis (1 µm) were continuously run during acquisitions. The graph shows the quantification of double-positive particles. Mean and error bars derive from three independent experiments. Significance antiHER2 vs CD81-GFP EVs is ***P < 0.001. C Sandwich designed for the AlphaLISA competitive assay. CD81-GFP and antiHER2 EVs were tested for competition with HER2-DDK. The graph shows the measured alpha counts normalized to the GFP-positive EV population as calculated by NTA and imaging flow cytometry. Mean and SD derive from three independent experiments (significance is ****P < 0.0001). D Representative western blotting of recombinant EVs immunoprecipitation with HER2-DDK or anti-GFP antibody in serum-free DMEM. AntiHER2 GFP-positive fusion proteins are enclosed in the yellow box above the antibody heavy chains (black arrow). Controls of beads flow through with HER2-DDK (*) or anti-GFP antibody (**) are shown on the right, indicating saturation of the beads’ surface to avoid non-specific binding. The graph shows the densitometric quantification of antiHER2 EVs captured by both HER2-DDK and anti-GFP Ab, with a competition effect of Trastuzumab. Mean and SD refer to two independent experiments
Article Snippet: EV samples in PBS were labeled with 1 μg/ml (final concentration) of Cell Mask Deep Red (CMDR, Invitrogen) in ratio 1:1 (v/v) and incubated at RT for 20 min. Then, samples were diluted in PBS to obtain a final concentration lower than 10^10 objects/ml before acquisition.
Techniques: Western Blot, Imaging, Flow Cytometry, Labeling, Recombinant, Immunoprecipitation, Binding Assay
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