Journal: Scientific Reports

Article Title: Imaging of angiogenesis of human umbilical vein endothelial cells by uptake of exosomes secreted from hepatocellular carcinoma cells

doi: 10.1038/s41598-018-24563-0

Figure Lengend Snippet: The isolation, characterization, and observation of exosomes secreted from HepG2 cells. ( a ) The exosomes secreted from HepG2 cells (HepG2-exosomes) isolated by the ExoQuick-TC kit in the bottom of the tubes were found as white pellets. Both white arrows show the exosomes. ( b ) An image of transmission electron microscopy of exosomes secreted from HepG2. ( c ) The size distribution, average size and zeta potential of HepG2-exosomes in distilled water. ( d–h ) The expression of marker molecules such as CD63, CD81, NKG2D and HSP70 on the surface of HepG2-exosomes. ( i ) The protein concentration of exosomes secreted from HepG2, as determined by the BCA method. ( j–m ) The exosomes in HepG2 cells were detected by using a FITC-labeled anti-human CD63 mouse monoclonal antibody. The morphology of the cells ( j ), nuclei labeled with Hoechst33342 ( k ), exosomes labeled with FITC-labeled anti-human CD63 mouse monoclonal antibody exist in HepG2 cells ( l ) and the merged images of the nuclei and exosomes ( m ) are shown. White arrows show the exosomes. ( n,o ) Three-dimensional images of nuclei labeled with Hoechst33342 ( n ) and exosomes bound to the FITC-labeled anti-human CD63 mouse monoclonal antibody ( o ) found in HepG2 cells. White arrows show the exosomes. These figures were obtained using superresolution structured illumination microscopy (SR-SIM, Carl Zeiss).

Article Snippet: After washing the samples with the culture medium three times, they were observed by superresolution structured illumination microscopy (SR-SIM (ELYRA), Carl Zeiss Co., Ltd.).

Techniques: Isolation, Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Expressing, Marker, Protein Concentration, Labeling, Microscopy

Journal: American Journal of Physiology - Cell Physiology

Article Title: Assessment of roles for the Rho-specific guanine nucleotide dissociation inhibitor Ly-GDI in platelet function: a spatial systems approach

doi: 10.1152/ajpcell.00274.2016

Figure Lengend Snippet: Ly-GDI is phosphorylated at PKC substrate motifs following platelet activation and colocalizes with PKC in adherent platelets. A: FpClass predicted interaction partners for Ly-GDI (ARHGDIB) visualized by GeNets. B: Pathway Commons neighborhoods of curated Ly-GDI (ARHGDIB) interaction partners and regulatory proteins. Directed edges (arrows) indicate signaling steps and undirected edges indicate protein interactions. C: ChiBE “detailed view” of the specific phosphorylations of Ly-GDI by PKCα (PRKCA) as well as Src and Syk as curated by PhosphoSitePlus. D: washed human platelets (1 × 109/ml) were stimulated with CRP or vehicle alone (10 min, 37°C) before collection into IP buffer and immunocapture with Ly-GDI antibodies or nonspecific rabbit IgGs. After IP, protein A/G eluates were analyzed for PKC substrate phosphorylation and total Ly-GDI protein capture by Western blotting (WB). For WB panels, tick marks indicate position of 28-kDa molecular mass marker. E: replicate samples of washed human platelets (2 × 107/ml) were spread on glass coverslips coated with fibrinogen before fixation and staining for RhoGDI or Ly-GDI (green) together with PKC (red) and visualized by conventional fluorescence microscopy (scale bar, 10 μm). Pearson’s correlation reveals significantly increased colocalization of Ly-GDI and PKC relative to RhoGDI and PKC (indicated by *). F: wide-field (scale bar, 5 μm) and magnified (scale bar, 2 μm) superresolution structured illumination microscopy imaging of adherent platelets stained for Ly-GDI (green) and PKC (red).

Article Snippet: Adherent platelets were also imaged using superresolution structured illumination microscopy (SR-SIM) with a Zeiss ×100 oil immersion 1.46 NA lens on a Zeiss Elyra PS.1 microscope as previously described ( 8 ).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Marker, Staining, Fluorescence, Microscopy, Imaging