Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 1 High expression of Nectin4 and FAP in a variety of cancers. (A) Expression of Nectin4 and FAP in glioma, liposarcoma, and leiomyosarcoma was assessed by IHC. (B) Expression of Nectin4 on lung-metastasized esophageal cancer, lung-metastasized liver cancer, and bone-metastasized triple-negative breast cancer (TNBC). Also see Supplementary Figure 3. Nectin4 is mainly located in the membrane (strongly positive) and cytoplasm (weakly positive) of cancer cells; FAP is mainly located in the membrane (strongly positive) and cytoplasm (weakly positive) of stromal cells, shown in brown.

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Expressing, Membrane

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 2 CAR structure and characterization of Nectin4-7.19 CAR-T cells. (A) Schematic illustration of Nectin4 CAR and Nectin4-7.19 CAR lentiviral vector. LTR: long terminal repeats; SP: CD8 signal peptide; TM: transmembrane region; P2A: 2A polypeptide element. (B) CAR expression in Nectin4 CAR-T and Nectin4-7.19 CAR-T cells was measured by flow cytometry. UTD indicates the untransduced T cells as a negative control. (C) Relative CAR expression in CD4+ and CD8+ T subsets. (D) Representative CAR-T cell phenotyping plot based on CD45RA and CCR7 in CD4+

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Plasmid Preparation, Expressing, Cytometry, Negative Control

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 3 Efficient cytotoxicity of Nectin4-7.19 CAR-T cells in vitro. (A) Expression of Nectin4 on a panel of cancer cell lines. (B) Cytotoxicity of Nectin4 CAR-T cells against ABC-1, HT1376, and MDA-MB-453 cells was detected by xCELLigence RTCA label-free technology. The left panel compares the cytotoxicity between Nectin4 CAR-T and CD19 CAR-T cells against target cells at an Effect/Target ratio of 10:1; the right panel shows the killing efficacy of Nectin4 CAR-T cells at different Effect/Target ratios. Arrows refer to the addition of CAR-T cells. The y-axis is the normalized cell index generated by the RTCA software and displayed in real time to reflect the vitality of tumor cells. The x-axis is the time of cell culture in hours. (C) Nectin4 and GFP expression in Luc. ABC-1 cells transfected with lentivirus encoding the Luciferase-T2A-GFP gene. (D) Quantified data on the specific lytic levels of Nectin4 CAR-T and Nectin4-7.19 CAR-T cells against Luc. ABC-1 cells were assessed by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. UTD served as a negative control. (E) Expression level of immune checkpoints was detected by flow cytometry after co-culture of Nectin4 CAR-T or Nectin4-7.19 CAR-T cells with ABC-1 cells at an Effect/Target ratio of 1:1 for 5 days. Data represent the mean ± SD of three independent experiments; ns, no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001, t-test.

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: In Vitro, Expressing, Generated, Software, Cell Culture, Transfection, Luciferase, Negative Control, Cytometry, Co-Culture Assay

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 4 Therapeutic effect of Nectin4 mCAR-T cells on metastatic colorectal cancer in fully immune-competent mice. (A) The murine CAR construct was inserted upstream of an IRES-GFP marker in the MSCV retroviral plasmid pMIGR1. (B) mCAR expression of Nectin4 mCAR-T cells transfected with pMIGR1-mCAR-IRES-GFP retroviral particles. mUTD indicates the untransduced mouse T cells. (C) Nectin4 and GFP expression of Luc. MC38 cells and hNectin4-Luc. MC38 cells. (D) Quantified data on the specific lytic levels of Nectin4 mCAR-T cells against Luc. MC38 or hNectin4-Luc. MC38 cells were assessed by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. ***p < 0.001, t-test. (E) Secretion of IFN-g in CD4+ and CD8+ T subsets was assessed by flow cytometry after co-culture of Nectin4 mCAR-T cells or mUTD with hNectin4-Luc. MC38 cells for 12 h. ***p < 0.001, t-test. (F, G) C57BL/6 mice were s.c. inoculated with 1 × 106 hNectin4-Luc. MC38 cells on Day 0 and injected i.v. with 5.0 × 105 to 5.0 × 106 Nectin4 mCAR-T cells on Day 10. A total of 5.0 × 106 mUTD served as a negative control (N = 6 mice per group). Solid lines represent each individual mouse (F). Kaplan–Meier survival curve is shown in (G). p-values of log-rank tests were as follows: p = 0.35 (mUTD vs. 0.5 × 106 Nectin4 mCAR-T); p = 0.09 (mUTD vs. 1.5 × 106 Nectin4 mCAR-T); p = 0.0012 (mUTD vs. 5.0 × 106

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Construct, Marker, Retroviral, Plasmid Preparation, Expressing, Transfection, Luciferase, In Vitro, Cytometry, Co-Culture Assay, Injection, Negative Control

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 5 Significant anti-tumor effect of Nectin4-7.19 CAR-T therapy on metastatic lung cancer without on-target off-tumor toxicity. (A) NSG mice were i.v. inoculated with 1.0 × 106 Luc. ABC-1 cells on Day 0 and received an administration of 3 × 106 Nectin4-7.19 CAR-T cells on Day 7 (N = 3 mice per group). Mice treated with the same dosage of UTD cells served as a negative control. (B–D) Tumor xenografts were monitored via bioluminescence imaging. Representative bioluminescence images of three independent experiments are shown in (B); bioluminescence kinetics are shown in (C); solid lines represent each individual mouse. Kaplan–Meier survival curve is shown in (D), p = 0.0246 (Nectin4-7.19 CAR-T vs. UTD), N = 3, log-rank test. (E) Body weight of mice since the tumor inoculation.

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Negative Control, Imaging

Journal: Frontiers in immunology

Article Title: Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

doi: 10.3389/fimmu.2022.958082

Figure Lengend Snippet: FIGURE 6 Synergistic effect of Nectin4-7.19 CAR-T with FAP-12 CAR-T therapy on metastatic lung cancer mouse model. (A) Schematic illustration of FAP CAR and FAP-12 CAR lentiviral vector. LS: leader signal. (B) CAR expression on FAP CAR-T and FAP-12 CAR-T cells. (C) Expression of CD45RA and CD45RO in CD4+ or CD8+ T subset to assess the subtypes of T cells. (D) 293T cells were transduced with lentivirus encoding the human FAP-Firefly-Luciferase-GFP gene or the murine FAP-Firefly-Luciferase-GFP gene to generate hFAP-Luc. 293T and mFAP-Luc. 293T cells, respectively. Expression of GFP was measured by flow cytometry. 293T cells served as negative controls. (E) Quantified data on the specific lytic levels of FAP CAR-T cells against hFAP-Luc. 293T and mFAP-Luc. 293T cells were assessed by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. UTD served as a negative control. (F) Cytotoxicity of FAP CAR-T and FAP-12 CAR-T cells was detected at an Effect/Target ratio of 10:1 by xCELLigence RTCA label-free technology. (G) Specific lysis of FAP CAR-T and FAP-12 CAR-T cells against hFAP- Luc. 293T was detected by luciferase bio-luminescence technique at different Effect/Target ratios in vitro. (H) NSG mice were inoculated with 1.0 × 106 Luc. ABC-1 cells i.v. on Day 0 and received an administration of 2 × 106 FAP-12 CAR-T cells, 2 × 106 Nectin4-7.19 CAR-T cells, or an admixture of 1 × 106 Nectin4-7.19 CAR-T cells and 1 × 106 FAP-12 CAR-T cells on Day 3 (N = 3 mice per group). A total of 2.0 × 106 UTD served as a negative control. (I) Tumor xenografts were monitored via bioluminescence imaging. Representative bioluminescence images of three independent experiments in each group of mice were shown. (J) Bioluminescence kinetics of the tumor growth in the model. (K) Kaplan– Meier survival curve. p-values of log-rank tests were as follows: p = 0.0246 (Nectin4-7.19+FAP-12 vs. UTD); p = 0.0246 (Nectin4-7.19+FAP-12 vs. FAP-12); p = 0.1161 (Nectin4-7.19+FAP-12 vs. Nectin4-7.19), N = 3. (L) Body weight of mice since the tumor inoculation. Data represent the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, t-test.

Article Snippet: Expression of Nectin4 on the surface of tumor cells was detected by anti-human Nectin4 Alexa Fluor® 647-conjugated antibody (Clone #337516, R&D Systems, USA); Nectin4 CAR and Nectin4-7.19 CAR expression was detected by the fusion protein of Nectin4 extracellular domain with streptavidin (Nectin4-streptavidin), and then followed by the antistreptavidin antibody with PE fluorescein (Clone #3A20.2).

Techniques: Plasmid Preparation, Expressing, Transduction, Luciferase, Cytometry, In Vitro, Negative Control, Lysis, Imaging

Journal: Virology

Article Title: Canine distemper virus with the intact C protein has the potential to replicate in human epithelial cells by using human nectin4 as a receptor.

doi: 10.1016/j.virol.2012.10.033

Figure Lengend Snippet: Fig. 1. Infection of the parental, nectin4-expressing, and SLAM-expressing Vero cells with wild-type CDV strains. (A) Vero/hNectin4 (left panel) and Vero/dNectin4 (right panel) cells were stained with a goat anti-human nectin4 polyclonal antibody (gray empty profile) or a control goat IgG (filled black profile), followed by staining with Alexa Fluor 488-conjugated anti-goat IgG. (B) Vero, Vero/ hNectin4, Vero/dNectin4, Vero/hSLAM, and Vero.DogSLAMtag cells were infected with wild-type CDV strains (Ac96I-VDS, 82Con, 55L, M24Cr, and Th12) or mock- infected. At 48 h (Vero, Vero/hNectin4, Vero/dNectin4, and Vero/hSLAM) or 24 h (Vero.DogSLAMtag) post-infection, the cells were stained with the Giemsa solu- tion, and observed under a phase-contrast microscope. (C) Replication kinetics of Ac96I. Vero/hNectin4, Vero/dNectin4, and parental Vero cells were infected with Ac96I at a MOI of 0.01. At various time intervals post-infection, the virus titers were determined by plaque assays.

Article Snippet: The cell surface expression of nectin4 was analyzed using a goat anti-human nectin4 polyclonal antibody (R&D Systems, Minneapolis, MN) as the primary antibody and Alexa Fluor 488-conjugated anti-goat IgG (Molecular probes, Eugene, Oregon) as the secondary antibody.

Techniques: Infection, Expressing, Staining, Control, Microscopy, Virus

Journal: Virology

Article Title: Canine distemper virus with the intact C protein has the potential to replicate in human epithelial cells by using human nectin4 as a receptor.

doi: 10.1016/j.virol.2012.10.033

Figure Lengend Snippet: Fig. 2. Amino acid sequence comparison of the V domains of human, dog, and mouse nectin4. Dots indicate identical residues to those of human nectin4.

Article Snippet: The cell surface expression of nectin4 was analyzed using a goat anti-human nectin4 polyclonal antibody (R&D Systems, Minneapolis, MN) as the primary antibody and Alexa Fluor 488-conjugated anti-goat IgG (Molecular probes, Eugene, Oregon) as the secondary antibody.

Techniques: Sequencing, Comparison

Journal: Virology

Article Title: Canine distemper virus with the intact C protein has the potential to replicate in human epithelial cells by using human nectin4 as a receptor.

doi: 10.1016/j.virol.2012.10.033

Figure Lengend Snippet: Fig. 4. Replication kinetics in NCI-H358 cells. (A) NCI-H358 cells were stained with a goat anti-human nectin4 polyclonal antibody (gray empty profile) or a control goat IgG (filled black profile), followed by staining with Alexa Fluor 488-conjugated anti-goat IgG. (B) NCI-H358 cells were infected with the Ac96I-VDS, 82Con, 55 L, M24Cr, or Th12 CDV strains at a MOI of 0.01. At 5 days post-infection, the virus titers were determined by plaque assays. (C, D) NCI-H358 (C) and II-18 (D) cells were infected with Ac96I-VDS or Ac96I-H358 at a MOI of 0.01. At 1, 3, 5, and 7 days post-infection, the virus titers were determined by plaque assays.

Article Snippet: The cell surface expression of nectin4 was analyzed using a goat anti-human nectin4 polyclonal antibody (R&D Systems, Minneapolis, MN) as the primary antibody and Alexa Fluor 488-conjugated anti-goat IgG (Molecular probes, Eugene, Oregon) as the secondary antibody.

Techniques: Staining, Control, Infection, Virus

Journal: Fujita Medical Journal

Article Title: Increased levels of nectin-4 as a serological marker for pre-eclampsia

doi: 10.20407/fmj.2022-027

Figure Lengend Snippet: Serum concentrations of nectin-4 measured by ELISA. A. High serum nectin-4 concentrations were present in serum samples from patients with pre-eclampsia. These levels were compared between the sera from cases with an uncomplicated normotensive pregnancy (left), pre-eclampsia (center) and unexplained fetal growth retardation without hypertension (right). The boxes indicate the 25th and 75th percentiles, and bands near the middle indicate median values. The bars indicate 1.5 interquartile ranges with the outliers specifically marked. B. Correlation between serum nectin-4 levels and disease onset. C. Correlation between nectin-4 levels and gestational week in normotensive uncomplicated pregnancies throughout gestation. D. Correlation between the corrected serum nectin-4 levels, divided by the mean value of serum nectin-4 from uncomplicated normotensive controls matched for gestational week.

Article Snippet: The serum nectin-4 concentrations were measured using a commercially available Quantikine ® ELISA kit, Human Nectin-4 Immunoassay (R&D Systems, Minneapolis, MN), in accordance with the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Gene therapy

Article Title: Measles virus selectively blind to signaling lymphocyte activation molecule as a novel oncolytic virus for breast cancer treatment.

doi: 10.1038/gt.2012.44

Figure Lengend Snippet: Figure 2. PVRL4 and not CD46 is involved in the infection of breast cancer cell lines with rMV-SLAMblind. (a) Surface expression of CD46 and PVRL4 in breast cancer cell lines analyzed by flow cytometry. (Left) Cells incubated with anti-CD46 mouse MAb (gray histogram) or isotype control (white histogram) followed by incubation with Alexa-488-conjugated goat anti-mouse antibody. (Right) Cells incubated with anti- PVRL4 goat polyclonal antibody (gray histogram) or isotype control (white histogram) followed by incubation with Alexa-488-conjugated rabbit anti-goat antibody. (b) Cells were pretreated with anti-CD46 or anti-PVRL4 antibody and infected with rMV-EGFP, rMV-EGFP-SLAMblind or rMV-Edmonston at an MOI of 0.1. Cells were incubated in medium with or without antibody and were photographed at 2 (Vero) or 3 d.p.i. (the others). Cells infected with rMV-Edmonston were immunostained using anti-N rabbit polyclonal antibody. Magnification: 100. (c) CHO- K1 cells were transfected with pCAG-hCD46 or the empty vector (pCAGGS). After 2 days, they were infected with rMV, rMV-SLAMblind or rMV- Edmonston at an MOI of 1 and incubated in the presence of fusion inhibitory peptide. At 2 d.p.i., cells were fixed and immunostained using anti-N MAb and the number of infected cells was counted. Infectivity in cells transfected with pCAGGS was set as 100%. Error bars indicate s.e. *Po0.001 versus pCAGGS. (d) BHK cells were transfected with pCAG-hPVRL4 or pCAGGS. After two days, they were infected with rMV, rMV- SLAMblind or rMV-Edmonston at an MOI of 0.1 and incubated in the presence of fusion inhibitory peptide. At 2 d.p.i., infectivity was determined as described in (c). *Po0.001 versus pCAGGS.

Article Snippet: Infection inhibition assay using antibodies Monolayers of MCF7, MDA-MB-453, SKBR3 and Vero cells in 96-well plates were pretreated for 1 h at 37 1C with medium containing 10mg ml 1 of anti-human CD46 MAb (clone M177; HyCult Biotechnology, Uden, The Netherlands)50 or anti-human PVRL4 goat polyclonal antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Infection, Expressing, Cytometry, Incubation, Control, Transfection, Plasmid Preparation

Journal: Gene therapy

Article Title: Measles virus selectively blind to signaling lymphocyte activation molecule as a novel oncolytic virus for breast cancer treatment.

doi: 10.1038/gt.2012.44

Figure Lengend Snippet: Figure 3. Infection of CD46-positive normal human cells. (a) Surface expression of CD46, PVRL4 and SLAM in NHDFs analyzed by flow cytometry as described in Figures 1c and 2a. (b, c) NHDFs were infected with rMV-SLAMblind or rMV-Edmonston at an MOI of 1. (b) Cells fixed and immunostained with anti-N MAb at 3 d.p.i. Magnification: 100. (c) Cell viability measured at each time point by WST-1 assay.

Article Snippet: Infection inhibition assay using antibodies Monolayers of MCF7, MDA-MB-453, SKBR3 and Vero cells in 96-well plates were pretreated for 1 h at 37 1C with medium containing 10mg ml 1 of anti-human CD46 MAb (clone M177; HyCult Biotechnology, Uden, The Netherlands)50 or anti-human PVRL4 goat polyclonal antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Infection, Expressing, Cytometry, WST-1 Assay

Journal: Virology

Article Title: The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread.

doi: 10.1016/j.virol.2014.02.014

Figure Lengend Snippet: Fig. 1. Amino acid sequences of all human PVRL molecules and dog PVRL4 in the V domain. (A) Sequence alignment of the V domain from human PVRL1 to PVRL4, human PVR, and dog PVRL4. Residues having similarity are shaded (dark shading represents identical amino acid residues; dark gray shading represents residues with 80–100% identity, light gray shading represents residues with 60–80% identity, no shading represents residues with less than 60% identity). The consensus FPxG amino acid sequence is boxed in red. Pairwise protein alignments were performed using Geneious sequence alignment software (Drumond et al., 2010). (B) Schematic diagrams of the wild-type and chimeric dog PVRL4 and human PVRL1 proteins (dog.PVRL4, dog.PVRL4/VhPVRL1, hPVRL1 and hPVRL1/Vdog.

Article Snippet: Briefly, cells were washed with PBS, non-enzymatically dissociated and subsequently blocked in 2.5 μg of normal human IgG (R&D Systems) for 10 min on ice followed by the addition of 10 μl of either PE-conjugated monoclonal antibody against human PVRL4 27-351 (R&D Systems FAB2659P) or PE-conjugated mouse IgG2B isotype control (R&D Systems IC0041P), and APC-conjugated mouse monoclonal antibody against human PVRL1 31-334 (R&D Systems FAB2880A) or APC-conjugated mouse IgG2A isotype control (R&D Systems IC003A) antibodies for 45 min on ice.

Techniques: Sequencing, Software

Journal: Virology

Article Title: The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread.

doi: 10.1016/j.virol.2014.02.014

Figure Lengend Snippet: Fig. 2. Cell surface expression of chimeric PVRL molecules (dog.PVRL4, dog.PVRL4/VhPVRL1 and hPVRL1/Vdog.PVRL4). The flow cytometric histograms depict the cell surface expression profile of PVRL4 and PVRL1 in the corresponding cell line (isotype ctrl antibody, IgG – shaded; PVRL4 antibody, PVRL4 – blue; PVRL1 antibody, PVRL1 – green). Vero stable cell lines were incubated with either a phycoerythrin (PE)-conjugated mouse monoclonal antibody specific for human PVRL4, a PE-conjugated mouse IgG2a control antibody, an allophycocyanin (APC)-conjugated mouse monoclonal antibody specific for human PVRL1 or an APC-conjugated mouse IgG2B control antibody. The Y-axis represents cell counts and the X-axis represents fluorescence intensity (FL8: APC; FL2: PE).

Article Snippet: Briefly, cells were washed with PBS, non-enzymatically dissociated and subsequently blocked in 2.5 μg of normal human IgG (R&D Systems) for 10 min on ice followed by the addition of 10 μl of either PE-conjugated monoclonal antibody against human PVRL4 27-351 (R&D Systems FAB2659P) or PE-conjugated mouse IgG2B isotype control (R&D Systems IC0041P), and APC-conjugated mouse monoclonal antibody against human PVRL1 31-334 (R&D Systems FAB2880A) or APC-conjugated mouse IgG2A isotype control (R&D Systems IC003A) antibodies for 45 min on ice.

Techniques: Expressing, Stable Transfection, Incubation, Control

Journal: Virology

Article Title: The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread.

doi: 10.1016/j.virol.2014.02.014

Figure Lengend Snippet: Fig. 3. CDV 5804PeH replication in wild-type, chimeric dog PVRL4 and human PVRL1-expressing Vero stable cell lines. Vero.dog.PVRL4, Vero.dog.PVRL4/VhPVRL1, Vero. hPVRL1/Vdog.PVRL4, and Vero cells were seeded on twelve-well plate (1 105 cells/well) 1 day before infection with CDV 5804PeH at an MOI of 0.5. (A) Fluorescence images were captured three days post infection and overlaid with phase contrast images to visualize the extent of virus replication (GFP) and syncytia formation. (B) CDV-infected cells were harvested at 3 days post-infection and titered on Vero.dog.SLAM cells. Data are the means from three independent experiments, and error bars represent the SEM. Analysis of variance (ANOVA) was performed to identify statistically significant differences in CDV 5804PeH titrations. P values below 0.05 were considered statistically significant.

Article Snippet: Briefly, cells were washed with PBS, non-enzymatically dissociated and subsequently blocked in 2.5 μg of normal human IgG (R&D Systems) for 10 min on ice followed by the addition of 10 μl of either PE-conjugated monoclonal antibody against human PVRL4 27-351 (R&D Systems FAB2659P) or PE-conjugated mouse IgG2B isotype control (R&D Systems IC0041P), and APC-conjugated mouse monoclonal antibody against human PVRL1 31-334 (R&D Systems FAB2880A) or APC-conjugated mouse IgG2A isotype control (R&D Systems IC003A) antibodies for 45 min on ice.

Techniques: Expressing, Stable Transfection, Infection, Fluorescence, Virus

Journal: Virology

Article Title: The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread.

doi: 10.1016/j.virol.2014.02.014

Figure Lengend Snippet: Fig. 4. Characterization of CDV glycoproteins-induced cell–cell fusion in wild-type, chimeric dog PVRL4 and human PVRL1-expressing Vero stable cell lines. Vero.dog.PVRL4, Vero.dog.PVRL4/VhPVRL1, Vero.hPVRL1/Vdog.PVRL4, Vero.dog.SLAM, and Vero cells were cotransfected with plasmid encoding CDV F together with CDV H or CDV HP493S/Y539A. At 48 h post transfection, cells were stained with Giemsa as described in Materials and methods. Light microscopy images were captured to visualize the extent of syncytia formation. Arrow heads indicate the location of representative syncytia in transfected cells.

Article Snippet: Briefly, cells were washed with PBS, non-enzymatically dissociated and subsequently blocked in 2.5 μg of normal human IgG (R&D Systems) for 10 min on ice followed by the addition of 10 μl of either PE-conjugated monoclonal antibody against human PVRL4 27-351 (R&D Systems FAB2659P) or PE-conjugated mouse IgG2B isotype control (R&D Systems IC0041P), and APC-conjugated mouse monoclonal antibody against human PVRL1 31-334 (R&D Systems FAB2880A) or APC-conjugated mouse IgG2A isotype control (R&D Systems IC003A) antibodies for 45 min on ice.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Transfection, Staining, Light Microscopy