Journal: Vaccines

Article Title: Protective CD8+ T Cell Response Induced by Modified Vaccinia Virus Ankara Delivering Ebola Virus Nucleoprotein

doi: 10.3390/vaccines10040533

Figure Lengend Snippet: Generation of recombinant MVA-EBOV viruses. ( A , B ) Schemes of the MVA genome with the intergenic insertion site 069R-070L ( A ) or the major deletions sites I-VI ( B ). Flank-1 and flank-2 refer to MVA DNA sequences targeting the intergenic site 069R-070L ( A ) or deletion site III ( B ) in the MVA genome for insertion of recombinant genes. MVA vector plasmids contain recombinant EBOV NP or GP gene sequences, respectively, under transcriptional control of the vaccinia virus promoter PmH5 and a marker gene sequence for transient expression of the fluorescent protein GFP ( A ) or mCherry ( B ). Short repetitive sequences of MVA DNA (del) served to remove the marker genes by intragenomic homologous recombination (marker gene deletion). ( C , D ) Multiple-step growth analysis of recombinant MVA-EBOV viruses. Growth of recombinant viruses MVA-EBOV-NP ( C ) or MVA-EBOV-GP ( D ) was monitored upon infection of chicken fibroblast cells ( C , E , F ); hpi: hours post infection. ( E , F ) Synthesis of recombinant EBOV NP ( E ) and EBOV GP ( F ) was tested by Western blot analysis using cell lysates and supernatants from infected CEF cells. Polypeptides were separated by SDS–PAGE and tested by immunoblotting using either EBOV NP- or GP-specific antibodies. Uninfected cells (mock) or non-recombinant MVA-infected cells (MVA) served as controls.

Article Snippet: Briefly, the MVA clonal isolate F6 [ , ] was used as virus starting material and propagated on primary chicken embryo fibroblasts (CEF) obtained from 10-day old embryonated SPF chicken eggs (Valo BioMedia GmbH, Osterholz-Scharmbeck, Germany).

Techniques: Recombinant, Plasmid Preparation, Control, Virus, Marker, Sequencing, Expressing, Homologous Recombination, Infection, Western Blot, SDS Page

Journal: Journal of Cellular and Molecular Medicine

Article Title: Targeting STAT3 enhances NDV‐induced immunogenic cell death in prostate cancer cells

doi: 10.1111/jcmm.15089

Figure Lengend Snippet: Apoptosis and immunogenic cell death were induced by NDV/FMW in prostate cancer cells. A, DU145 and PC‐3 cells were infected with 0.01 MOI NDV/FMW for 24, 48 and 72 h, viral yield was determined at the indicated times by diluting serially in DF1 cells. Values are mean ± SD from three independent tests. B, DU145 and PC‐3 cells were mock‐infected or infected with NDV/FMW (MOI = 1) for 12, 24 and 48 h. Cell viablity were assessed by the CCK‐8 assay. Values are mean ± SD. (n = 3, *** P < .001). C, Cells were treated the same as in (B). Cell death was quantified by trypan blue staining. Values are mean ± SD (n = 3, *** P < .001, n.s = not significant). D, DU145 and PC‐3 cells were mock‐infected or infected with 1 MOI NDV/FMW for 24 and 48 h. Apoptosis was determined by flow cytometry. The group of cells in Annexin V‐positive with PI‐negative quadrant and Annexin V/PI positive quadrant are illustrated. The data displayed are representative of three independent tests (* P < .05; ** P < .01 and *** P < .001). E, DU145 and PC‐3 cells were treated the same as in (B). IB was used to determine the expression of the cleaved‐PARP, Beclin‐1, p‐eIF2α and haemagglutinin‐neuraminidase (HN) protein. Using β‐actin as a loading control. Protein intensity was quantified with Image Lab and shown in the histogram. All IB experiments were made three times. F, DU145 and PC‐3 cells were mock‐infected or infected with 10 MOI NDV/FMW for 12, 24 and 48 h, cell‐free supernatants (concentrated) and whole cell lysates were collected for IB analysis of HMGB1 and HSP70/90. G, DU145 and PC‐3 cells were treated the same as in (B), and cell‐free supernatants were collected to measure the amounts of HMGB1 using an enzyme‐linked immunosorbent (ELISA) detection. Existing data are mean ± SD (n = 3, n.s = not significant, *** P < .01). (H) DU145 and PC‐3 cells were treated the same as in (D), extracellular ATP in cell‐free supernatants was evaluated by ATP estimation. Existing data are mean ± SD calculated from three independent experiments (* P < 0.05; ** P < .01, n.s = not significant). I, DU145 and PC‐3 cells were treated the same as in (D). The cells were assessed by immunofluorescence staining and analysed under a confocal laser‐scanning microscope. Mitoxantrine (MTX), an ICD inducer, was used as a positive control. Nucleus was stained by DAPI. NDV/FMW was detected based on HN protein. The fluorescence intensity was semi‐quantitatively determined by Image J, and the results were expressed by mean density (n = 3, * P < .05; *** P < .001, ns = not significant)

Article Snippet: Human prostate cancer cells PC‐3 and fibroblast cell line from Chicken embryo DF1 were purchased from the China Center for Type Culture Collection (Shanghai, People's Republic of China).

Techniques: Infection, CCK-8 Assay, Staining, Flow Cytometry, Expressing, Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Laser-Scanning Microscopy, Positive Control, Fluorescence