neongreen  (Cell Signaling Technology Inc)

Journal: Viruses

Article Title: The Role of the Tyrosine-Based Sorting Signals of the ORF3a Protein of SARS-CoV-2 in Intracellular Trafficking and Pathogenesis

doi: 10.3390/v17040522

Figure Lengend Snippet: Figure 3. The SARS-CoV-2 ORF3a is expressed in organelles of the secretory pathway and at the cell-plasma membrane. COS-7 cells were co-transfected with the empty pcDNA.3.1(+) vector or vectors expressing SARS-CoV-2 ORF3a-HA protein and vectors expressing markers for the rough endoplasmic reticulum (RER; ERmoxGFP), cis/medial Golgi (mNeonGreen-Giantin), trans Golgi network (TGN38 EGFP), and mitochondria (4xmts-mNeonGreen). In other cultures, COS-7 cells were

Article Snippet: To examine the co-localization of the ORF3a proteins with mitochondria, COS-7 cells were co-transfected with vectors expressing the ORF3a proteins and a vector expressing 4xmts-NeonGreen (mitochondria; Addgene, #98876).

Techniques: Clinical Proteomics, Membrane, Transfection, Plasmid Preparation, Expressing

Journal: Viruses

Article Title: The Role of the Tyrosine-Based Sorting Signals of the ORF3a Protein of SARS-CoV-2 in Intracellular Trafficking and Pathogenesis

doi: 10.3390/v17040522

Figure Lengend Snippet: Figure 4. The ORF3a-∆YxxΦ is not expressed at the cell-plasma membrane. HEK293 cells were transfected with the empty pcDNA.3.1(+) vector or a vector expressing the SARS-CoV-2 HA-ORF3a- [∆YxxΦ] protein section as in Figure 3. (A). Cells transfected with vectors expressing HA-ORF3a- [∆YxxΦ] and a vector expressing a marker for the rough endoplasmic reticulum (RER; ERmoxGFP) and immunostained with an anti-HA antibody. (B). Cells transfected with a vector expressing HA-ORF3a-[∆YxxΦ] and immunostained with antibodies against ERGIC-53 and HA. (C). Cells trans- fected with vectors expressing HA-ORF3a-[∆YxxΦ] and mNeonGreen-Giantin and immunostained with an anti-HA antibody. (D). Cells transfected with the vector expressing HA-ORF3a-[∆YxxΦ] and immunostained with antibodies against Golgin 97 and HA. (E). Cells transfected with vectors expressing HA-ORF3a-[∆YxxΦ] and TGN38 EGFP and immunostained with an anti-HA antibody. (F). Cells transfected with the vector expressing HA-ORF3a-[∆YxxΦ] and 4xmts-mNeonGreen and immunostained with antibodies against HA.

Article Snippet: To examine the co-localization of the ORF3a proteins with mitochondria, COS-7 cells were co-transfected with vectors expressing the ORF3a proteins and a vector expressing 4xmts-NeonGreen (mitochondria; Addgene, #98876).

Techniques: Clinical Proteomics, Membrane, Transfection, Plasmid Preparation, Expressing, Marker

Journal: bioRxiv

Article Title: Enhancing the efficiency of oncolytic vaccinia virus for ovarian cancer treatment

doi: 10.1101/2025.02.04.636413

Figure Lengend Snippet: A . Immunoblot analysis comparing the expression levels of the indicated viral proteins in HeLa cells infected with the indicated viruses (MOI of 1) at 4 and 8 hpi. Vinculin is the cell loading control and NeonGreen is only detected in ΔVFTK-NG and ΔVFTK-NG-GM-CSF infected cells. The experiment was repeated three times and a representative example is shown. B. Representative images of plaque formation by the indicated virus strains in BS-C-1 cells at 72 hpi. The graph represents quantitative analysis of plaque diameter measured in mm for the indicated viral strains. The three independent plaque assays are represented by the three different colours in the SuperPlot. Each dot represents one plaque, and the triangles represent the medians of all the plaques measured in each experiment. Data are represented as mean ± SD. One-way ANOVA was used to determine significance between all groups with Tukey multiple comparisons post-hoc test. C . Immunoblot analysis reveals increased PARP cleavage following infection of ID8 Trp53 -/- cells with ΔVF, ΔVFTK, ΔVFTK-NG, ΔVFTK-NG-GM-CSF compared to WR after 48 hours. F12 and GRB2 represent viral and cell loading controls respectively. The asterisk (*) indicates a non-specific band. Hpi = hours post infection. The experiment was repeated three times and a representative blot is shown.

Article Snippet: For immunohistochemistry (IHC), samples were stained for NeonGreen (1:200, Cell Signalling, #41236) and cleaved caspase 3 (1:250, Cell signalling, #9579S) antibodies, respectively.

Techniques: Western Blot, Expressing, Infection, Control, Virus

Journal: bioRxiv

Article Title: Enhancing the efficiency of oncolytic vaccinia virus for ovarian cancer treatment

doi: 10.1101/2025.02.04.636413

Figure Lengend Snippet: A. Schematic summarising the high throughput drug screening strategy. B. Representative images of ID8 Trp53 -/- cells infected with ΔVFTK-NG virus and treated with the indicated compounds belonging to the four categories. NeonGreen expression (green) indicates infected cells and DAPI was used to stain nuclei (blue). Cells were imaged using Opera Phenix Plus with 10x air objective. Scale bar = 200 μm. C. Graphical illustration of compound allocation in the four categories. D. Representative graphs from the secondary validation screen of selected primary screen hits at 1μM (omipalisib, erlotinib and vinorelbine). The model represents an ideal compound and DMSO is the negative control. The purple dashed line represents the time at which ΔVFTK-NG virus (MOI 0.5) was added (16 hours post cell seeding). Statistical analysis was done by comparing uninfected against infected cell confluency at 20, 60 and 90 hours post cell seeding using Student’s t-test. Error bars represent standard deviation (SD). E. List of hit compounds after the secondary validation screen.

Article Snippet: For immunohistochemistry (IHC), samples were stained for NeonGreen (1:200, Cell Signalling, #41236) and cleaved caspase 3 (1:250, Cell signalling, #9579S) antibodies, respectively.

Techniques: High Throughput Screening Assay, Infection, Virus, Expressing, Staining, Negative Control, Standard Deviation

Journal: bioRxiv

Article Title: Enhancing the efficiency of oncolytic vaccinia virus for ovarian cancer treatment

doi: 10.1101/2025.02.04.636413

Figure Lengend Snippet: A. Schematic representation of the experimental design of the distribution study. Mice were injected IP with ID8 Trp53 -/- cells on day 0 and received their IP injection on day 28 and the combination groups received their second component (vinorelbine or virus) on day 29. Heat inactivated (HI) virus (ΔVFTK-NG-GM-CSFi) is the negative control. B. Quantification of viral DNA in omental tumour, liver and spleen measured by qPCR and expressed relative to viral DNA levels of the liver sample in the HI virus group. Error bars represent mean ± SD. C. Representative immunohistochemical images of the distribution of cleaved caspase 3 and NeonGreen in omental tumour, liver and spleen harvested from a mouse in Group 4. D. Representative immunohistochemical images of the distribution of cleaved caspase 3 and NeonGreen in omental tumours from mice in Groups 1, 3 and 4. E. Schematic representation of the experimental design of the vaccinia-vinorelbine combination study. Mice were injected with ID8 Trp53 -/- cells on day 0 and started receiving their IP treatment injections on day 21. Mice allocated to single treatment groups received their inoculations on days 21, 28 and 35 and those allocated to the combination groups received vinorelbine and ΔVFTK-NG-GM-CSF 24 hours apart. Heat inactivated (HI) ΔVFTK-NG-GM-CSFi virus was the negative control. F. Kaplan-Meier survival curve showing survival data for each group analysed by log-rank test. One mice belonging to group 2 was excluded from the analysis as after IP injection of ID8 Trp53 -/- cells omental tumour failed to form. The analysis is from the combination of two survival experiments following the same protocols.

Article Snippet: For immunohistochemistry (IHC), samples were stained for NeonGreen (1:200, Cell Signalling, #41236) and cleaved caspase 3 (1:250, Cell signalling, #9579S) antibodies, respectively.

Techniques: Injection, Virus, Negative Control, Immunohistochemical staining