Journal: The Journal of Infectious Diseases

Article Title: Middle East Respiratory Syndrome Coronavirus Efficiently Infects Human Primary T Lymphocytes and Activates the Extrinsic and Intrinsic Apoptosis Pathways

doi: 10.1093/infdis/jiv380

Figure Lengend Snippet: Middle East respiratory syndrome coronavirus (MERS-CoV) differentially infects subsets of human peripheral blood mononuclear cells (PBMCs). A , Human PBMCs were infected with MERS-CoV at 2 50% tissue culture infective doses per cell. At 24 hours after infection, infected cells were fixed with 4% paraformaldehyde (PA) and immunolabeled for detection of cell surface markers and MERS-CoV nucleoprotein (NP). The shaded curve and the solid line represent MERS-CoV NP expression from mock-infected and MERS-CoV–infected cells, respectively. The summary panel at the right represents the average percentage of infected cells from 3 different donors. B , Uninfected human PBMCs were fixed with 4% PA and immunolabeled for detection of surface DPP4 expression. The shaded curve and the solid line represented isotype and DPP4-specific staining, respectively. The summary panel at the right represents the average mean fluorescent intensity (MFI) from 3 different donors. In all panels, bars and error bars represented means and standard deviations, respectively. Statistical analyses were performed using the Student t test. * P < .001. Abbreviation: NK, natural killer.

Article Snippet: DPP4 antibody was obtained from R&D Systems.

Techniques: Infection, Immunolabeling, Expressing, Staining

Journal: The Journal of Infectious Diseases

Article Title: Middle East Respiratory Syndrome Coronavirus Efficiently Infects Human Primary T Lymphocytes and Activates the Extrinsic and Intrinsic Apoptosis Pathways

doi: 10.1093/infdis/jiv380

Figure Lengend Snippet: Middle East respiratory syndrome coronavirus (MERS-CoV) efficiently infects CD4 + and CD8 + T cells and downregulates surface dipeptidyl peptidase 4 (DPP4) in the infected cells. A , T cells were infected with MERS-CoV and severe acute respiratory syndrome CoV (SARS-CoV) at 2 50% tissue culture infective doses (TCID 50 ) per cell. Cells were fixed at 24 hours after infection and immunolabeled for detection of expression of MERS-CoV NP or SARS-CoV NP. The shaded curve and the solid line represent virus NP expression from mock-infected and MERS/SARS-CoV–infected cells, respectively. B , T cells were infected with MERS-CoV at 2 TCID 50 per cell. Cells were fixed at 24 hours after infection and immunolabeled for detection of CD4 or CD8 and MERS-CoV NP expression. The shaded curve and the solid line represent NP expression from mock-infected and MERS-CoV–infected cells, respectively. C , Uninfected T cells were fixed and immunolabeled for detection of CD4 or CD8 and surface DPP4 expression. The shaded curve and the solid line represent isotype and DPP4-specific staining, respectively. D , T cells were infected with MERS-CoV at 2 TCID 50 per cell. Cells were fixed at 24 hours after infection and immunolabeled for detection of CD4 or CD8, DPP4, and MERS-CoV NP expression. Surface or total DPP4 was detected by labeling the cells with the DPP4 antibody before or after cell permeabilization, respectively. The shaded curve represents isotype staining of DPP4. The solid line and dotted line represent DPP4 staining from mock-infected and MERS-CoV–infected cells, respectively. The DPP4 mean fluorescence intensity (MFI) in infected cells was calculated on the basis of CD4 + /MERS-CoV NP–expressing or CD8 + /MERS-CoV NP–expressing double-positive T-cell data. The DPP4 MFI in mock-infected cells was calculated on the basis of CD4 + or CD8 + T-cell data. The summary panels at the right represent the average percentage of infected cells ( A and B ), MFI ( C ), or percentage of mock MFI ( D ) from 3 different donors. In all panels, bars and error bars represent means and standard deviations. Statistical analyses were performed using the Student t test. * P < .05.

Article Snippet: DPP4 antibody was obtained from R&D Systems.

Techniques: Infection, Immunolabeling, Expressing, Virus, Staining, Labeling, Fluorescence

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Antiangiogenic Effects and Therapeutic Targets of Azadirachta indica Leaf Extract in Endothelial Cells

doi: 10.1155/2012/303019

Figure Lengend Snippet: (b)

Article Snippet: DPP4 , 15.39 ± 4.21 , 15.78 ± 2.62 , Hs00175218_m1 , Aminopeptidase.

Techniques: Binding Assay, Protein Binding, Activity Assay, Ubiquitin Proteomics

Journal: Viruses

Article Title: Unveiling the Role of TMPRSS2 in the Proteolytic Activation of Pandemic and Zoonotic Influenza Viruses and Coronaviruses in Human Airway Cells

doi: 10.3390/v16111798

Figure Lengend Snippet: Activation of SARS-CoV and MERS-CoV S by TMPRSS2 and further host proteases. ( A , B ) HeLa cells were co-transfected with plasmids coding for MERS-CoV S ( A ) or SARS-CoV ( B ) with a C-terminal FLAG-tag and plasmids coding for human TMPRSS2 or HAT or murine TMPRSS4 or TMPRSS13. Cell lysates were analysed via SDS-PAGE at 48 h p.t. Western blot analysis was performed using an antibody targeting the C-terminal FLAG-tag. Tubulin was used as a loading control. The data shown are representative of three individual experiments. EV = empty vector. ( C , E ) HeLa cells co-expressing MERS-CoV ( C ) or SARS-CoV ( E ) S, as well as the respective receptors DPP4 or ACE2, were incubated for 48 h. After fixation, the cells were stained with a primary antibody targeting the C-terminal FLAG-tag of S and a fluorescence-coupled secondary antibody. DAPI was used to stain the nuclei. Representative images of three independent experiments are shown. The scale bar represents 100 µM. ( D , F ) Quantification of MERS-CoV S ( D ) or SARS-CoV ( F ) mediated cell–cell fusion. For each condition, ten randomly taken images were analysed by counting the nuclei per syncytium (with at least three nuclei). The data shown are the mean values + SEM of three independent experiments. Statistical significance was determined with a one-way ANOVA followed by Šídák’s multiple comparisons test. ns = not significant, ** = p < 0.01, **** = p < 0.0001.

Article Snippet: The expression vectors coding for SARS- or MERS-CoV S with a C-terminal FLAG-tag, pCMV3-MERS-Spike-cFLAG (VG40069-CF) and pCMV3-SARS-Spike-cFLAG (VG40150-CF), as well as a vector encoding human dipeptidyl peptidase 4 (DPP4) with a C-terminal HA-tag, pCMV3-hDPP4-cHA (HG10688-CY), were purchased from Sino Biological, Houston, TX, USA.

Techniques: Activation Assay, Transfection, FLAG-tag, SDS Page, Western Blot, Control, Plasmid Preparation, Expressing, Incubation, Staining, Fluorescence