Journal: Viruses
Article Title: Ebola Virus GP Activates Endothelial Cells via Host Cytoskeletal Signaling Factors
doi: 10.3390/v14010142
Figure Lengend Snippet: Ebola VLPs activate ECs and induce apoptosis. ( A ) The different VLPs preparations were purified from supernatants of transfected HEK293T cells and characterized using Western blotting with monoclonal antibodies (anti-GP, anti-VP40, and anti-NP). Supernatants from non-transfected cells were used as the negative control (Mock). Characterization with anti-GP indicated the absence of GP in VLP VP40/NP . ( B ) ECs were exposed to VLPs (10× dilution) or VLP VP40/NP (10× dilution) for 48 h. Following the treatment, cells were fixed and permeabilized, and the activation was detected by immunofluorescence analysis using a monoclonal antibody directed against ICAM-1. The cells were then stained with Hoechst 33358 for imaging (magnification, ×10). Human recombinant TNF-α and supernatants from non-transfected cells (mock) were used as the positive and negative controls, respectively. Only cells treated with VLPs and TNF-α induced the expression of ICAM-1. Unlike TNF-α, VLPs-mediated upregulation of ICAM-1 expression was associated with cytopathic effects on ECs. Neither VLP VP40/NP nor the mock control induced the upregulation of ICAM-1 expression. ( C ) The apoptotic rate was quantified using the fluorescence intensity of the nuclear staining with the TdT enzyme. Data represent the percent values of apoptotic cells derived from the normalization to the mock-treated and are expressed as mean ± SD ( n = 2) from two independent experiments. *** p = 0.0006 and **** p < 0.0001 compared to the mock. The apoptotic rate in VLPs-, TNF-α- and Actinomycin D (4 µg/mL)-treated cells were compared to that in the cells treated with media (mock), applying one-way ANOVA, followed by Dunnett’s multiple comparisons test. ( D ) Images of nuclear staining from VLPs- and TNF-α-treated cells are shown, with VLPs-treated cells exhibiting apoptotic morphology in comparison to the TNF-α-treated cells. ( E ) Close up images showing the morphology of ECs nuclei after treatment with VLPs and TNF-α. White arrows indicate disrupted and aggregated nuclei in contact with VLPs. VLPs: virus-like particles; ECs: endothelial cells; ICAM-1: intercellular adhesion molecules 1.
Article Snippet: To detect apoptosis in cultured ECs following appropriate treatments, the CF488 terminal dUTP nick end labeling (TUNEL) apoptosis detection kit (Biotium, Fremont, CA, USA) was used according to the manufacturer’s instructions.
Techniques: Purification, Transfection, Western Blot, Negative Control, Activation Assay, Immunofluorescence, Staining, Imaging, Recombinant, Expressing, Fluorescence, Derivative Assay, Comparison, Virus
