Journal: Insects

Article Title: Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

doi: 10.3390/insects15060375

Figure Lengend Snippet: Primers used for qPCR.

Article Snippet: The apoptosis rate of insect Sf9 cells at different time points after virus infection was analyzed using the Annexin V-Elab Fluor ® 647/PI apoptosis detection kit (Elabscience, Wuhan, China) and flow cytometry.

Techniques: Sequencing

Journal: Insects

Article Title: Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

doi: 10.3390/insects15060375

Figure Lengend Snippet: Insect Sf9 cells exhibit normal biological functions even when heterologous VSR is overexpressed. ( A ) The schematic diagram illustrates the construction of transient expression plasmids: (a) Plasmid pie1-p0, (b) Plasmid pie1-p19, and (c) Plasmid pie1-core. ( B ) Transfection of recombinant plasmids into Insect Sf9 cells for 48 h does not induce any morphological changes in their structure. ( C ) Western blot analysis using His-tag antibody confirms successful overexpression of VSR protein in Sf9 cells (48 h post-transfection), with α-tubulin serving as a control. ( D ) qPCR results reveal no significant alteration in the expression levels of apoptotic genes in normal insect Sf9 cells after 24–72 h of VSR protein overexpression. ( E ) Flow cytometry analysis demonstrates that the apoptosis rate of Sf9 cells remains unchanged across different groups following VSR protein overexpression (48 h post-transfection; Annexin V-647 and Propidium Iodide used as fluorescent dyes).

Article Snippet: The apoptosis rate of insect Sf9 cells at different time points after virus infection was analyzed using the Annexin V-Elab Fluor ® 647/PI apoptosis detection kit (Elabscience, Wuhan, China) and flow cytometry.

Techniques: Expressing, Plasmid Preparation, Transfection, Recombinant, Western Blot, Over Expression, Control, Flow Cytometry

Journal: Insects

Article Title: Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

doi: 10.3390/insects15060375

Figure Lengend Snippet: Schematic representation illustrating the generation of a recombinant baculovirus harboring the vsr gene, luciferase gene, and fluorescent protein gene. ( A ) Schematic diagram depicting the construction of recombinant plasmids pFBDM-vsr-polh-egfp driven by various promoters (vsr: p64-p0, p64-p19, p64-core, p10-p0, p10-p19, p10-core). ( B ) Schematic diagram outlining the assembly of recombinant plasmid pUCDM-p64-Rluc-polh-Fluc-polh-mCherry containing luciferase genes. ( C ) Illustration presenting E. coli Sw106 strain hosting MultiBacmid. ( D ) Diagram demonstrating the integration of the vsr gene, luciferase gene, and fluorescent protein gene into recombinant E. coli Sw106 MultiBacmid using Tn7 and Cre-loxP transposition technology. ( E ) Representation showcasing normal insect Sf9 cells. ( F ) Successful invasion of Sf9 cells by recombinant E. coli Sw106 facilitated by Invasin, resulting in rBacmid release. ( G ) Schematic diagram displaying the construction of high-expression recombinant baculoviruses carrying different vsr genes, luciferase genes, and fluorescent protein genes.

Article Snippet: The apoptosis rate of insect Sf9 cells at different time points after virus infection was analyzed using the Annexin V-Elab Fluor ® 647/PI apoptosis detection kit (Elabscience, Wuhan, China) and flow cytometry.

Techniques: Recombinant, Luciferase, Plasmid Preparation, Expressing

Journal: Insects

Article Title: Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

doi: 10.3390/insects15060375

Figure Lengend Snippet: The recombinant baculovirus carrying the target gene successfully infected insect Sf9 cells, and the expression of VSR protein in the cells was identified using Western blot. ( A ) After incubation of the recombinant baculovirus with normal insect Sf9 cells for 48 h (MOI = 1), fluorescence microscopy revealed the presence of both green and red fluorescence signals within the insect Sf9 cells, confirming successful infection by the recombinant virus and expression of the target protein (Bar = 50 µm). ( B ) After infecting insect Sf9 cells with the recombinant baculovirus, cell pellets were collected at various time points during infection. Subsequently, intracellular proteins were identified using Western blot analysis with His-tag antibody and α-tubulin antibody. The results demonstrated a significant upregulation of VSR in Sf9 cells between 48- and 96 h post-infection, with the highest expression level observed at 72 h post-infection. All the Western blot experiments were conducted as parallel experiments, and gel blots were processed simultaneously .

Article Snippet: The apoptosis rate of insect Sf9 cells at different time points after virus infection was analyzed using the Annexin V-Elab Fluor ® 647/PI apoptosis detection kit (Elabscience, Wuhan, China) and flow cytometry.

Techniques: Recombinant, Infection, Expressing, Western Blot, Incubation, Fluorescence, Microscopy, Virus

Journal: Insects

Article Title: Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

doi: 10.3390/insects15060375

Figure Lengend Snippet: Impact of early or later expression of heterologous VSR on the rate of virus-induced apoptosis in Sf9 insect cells at distinct time intervals. (*: p < 0.05; **: p < 0.01; Compared with the control group).

Article Snippet: The apoptosis rate of insect Sf9 cells at different time points after virus infection was analyzed using the Annexin V-Elab Fluor ® 647/PI apoptosis detection kit (Elabscience, Wuhan, China) and flow cytometry.

Techniques: Expressing, Virus, Control

Journal: Insects

Article Title: Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

doi: 10.3390/insects15060375

Figure Lengend Snippet: Repercussions of heterologous VSR on the regulation of apoptotic gene expression in virus-infected host Sf9 cells. ( A ) Heat map illustrating the impact of heterologous VSR on the regulation of apoptotic gene expression in Sf9 cells during viral infection, depicting early and late expression patterns. ( B ) Significance analysis of differential expression levels of host apoptotic genes at different time points during viral infection, with values above the green dotted line or below the yellow dotted line indicating statistical significance ( p < 0.05).

Article Snippet: The apoptosis rate of insect Sf9 cells at different time points after virus infection was analyzed using the Annexin V-Elab Fluor ® 647/PI apoptosis detection kit (Elabscience, Wuhan, China) and flow cytometry.

Techniques: Gene Expression, Virus, Infection, Expressing, Quantitative Proteomics

Journal: Insects

Article Title: Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

doi: 10.3390/insects15060375

Figure Lengend Snippet: Impact of heterologous VSR on protein expression efficiency in insect cell bioreactors. ( A ) The expression of early protein Renilla-luciferase in insect Sf9 cells was significantly enhanced by heterologous VSR. ( B ) The expression of late protein Firefly-luciferase in insect Sf9 cells was significantly increased upon the introduction of heterologous VSR.

Article Snippet: The apoptosis rate of insect Sf9 cells at different time points after virus infection was analyzed using the Annexin V-Elab Fluor ® 647/PI apoptosis detection kit (Elabscience, Wuhan, China) and flow cytometry.

Techniques: Expressing, Luciferase

Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: Endothelial cell tubule formation assay. BME cells (3 × 105/ml) were seeded onto type I collagen gels in medium supplemented with 10% calf serum. On the following day, the medium was aspirated and replaced with conditioned serum-free medium derived from cocultures of G8 myoblast monolayers and TA3wt (A) TA3sCD44 (B), TA3sCD44/MMP-9-CD44fp (C), or TA3sCD44/MMP-9v5 (D) cells. A total of 30 μg/ml of pan specific anti-TGF-β (E) or anti-bFGF (F) antibody were added to the TA3wt/G8-conditioned coculture medium prior to use in the assay. Tubules are indicated by arrows. Bar, 140 μm.

Article Snippet: Anti-MMP-9 antibody was from Oncogene (Cambridge, MA and Santa Cruz, CA) and anti-TGF-β1, TGF-β2, and TGF-β3 as well as pan-specific anti-TGF-β antibodies were from R & D Systems (Minneapolis, MN).

Techniques: Tube Formation Assay, Derivative Assay

Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: CD44-anchored MMP-9 activates TGF-β. (A) The ability of TA3 transfectant-G8 myoblast coculture media to stimulate TMLC luciferase activity is shown. Conditioned coculture media tested are indicated. (B) Pan-specific TGF-β antibody (30 μg/ml) or specific antibodies against TGF-β1 (100 ng/ml), TGF-β2(100ng/ml), or TGF-β3(100 ng/ml) were used to determine the activity of TGF-β isoforms in the coculture media. One unit of luciferase activity corresponds to the activity produced by 5 pg of purified human TGF-β1 (R & D).

Article Snippet: Anti-MMP-9 antibody was from Oncogene (Cambridge, MA and Santa Cruz, CA) and anti-TGF-β1, TGF-β2, and TGF-β3 as well as pan-specific anti-TGF-β antibodies were from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Luciferase, Activity Assay, Produced, Purification

Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: TGF-β is activated by purified MMP-9. (A) TMLC luciferase activity induced by concentrated COS cell supernatants containing the indicated latent TGF-β isoforms following incubation with the indicated purified AMPA-activated MMPs. Comparable amounts of latent TGF-β1, TGF-β2, and TGF-β3 were present in the supernatants as assessed by both Western blot analysis and TMLC-luciferase induction following heat treatment (80°C for 5min) (B) TMLC luciferase activity induced by affinity-purified TGF-β2 following incubation with the indicated purified activated MMPs. Activity is expressed in relative light units (RLU), in which 800 RLU corresponds to the luciferase activity generated by 1 pg of purified human TGF-β1 (R & D). (C) TGF-β is proteolytically cleaved by MMP-9 and MMP-2. Purified v5-tagged TGF-β2 (lane 1) was incubated with protein-A Sepharose-bound, AMPA-activated MMP-9 (lane 2), MMP-2 (lane 3), and MMP-3 (lane 4) for 90 min at 37°C. Following incubation, the supernatants were separated from the beads, subjected to SDS/12% PAGE, transferred to Hybond-C membranes, and blotted with anti-v5 antibody. (D) MMP-2/CD44 fusion protein expression promotes TGF-β activation in TA3sCD44 cells. TMLC luciferase assays were performed with serum-free coculture media from TA3sCD44 cells transiently transfected with the indicated cDNAs.

Article Snippet: Anti-MMP-9 antibody was from Oncogene (Cambridge, MA and Santa Cruz, CA) and anti-TGF-β1, TGF-β2, and TGF-β3 as well as pan-specific anti-TGF-β antibodies were from R & D Systems (Minneapolis, MN).

Techniques: Purification, Luciferase, Activity Assay, Incubation, Western Blot, Affinity Purification, Generated, Expressing, Activation Assay, Transfection

Journal: BioMed Research International

Article Title: Designation of a Novel DKK1 Multiepitope DNA Vaccine and Inhibition of Bone Loss in Collagen-Induced Arthritic Mice

doi: 10.1155/2015/765490

Figure Lengend Snippet: Designation of the DNA vaccine.(a) B cell epitope scanning of human DKK1 was performed with the software DNASTAR 7.1. (b) The affinity of epitopes was measured by indirect ELISA. (c) The separated epitopes were immunized BALB/c mice and the immunogenicity of epitopes was measured by sandwich ELISA. (d) The separated epitopes were injected to BALB/c mice for seven days. TRAP staining was performed to identify the mature osteoclasts. Magnification: 200x; data are expressed as the mean ± SEM.

Article Snippet: After blocking with 5% horse serum, the sections were incubated with a goat anti-human DKK1 antibody (5 μ g/mL, R&D Systems) overnight at 4°C.

Techniques: Software, Indirect ELISA, Immunopeptidomics, Sandwich ELISA, Injection, Staining

Journal: BioMed Research International

Article Title: Designation of a Novel DKK1 Multiepitope DNA Vaccine and Inhibition of Bone Loss in Collagen-Induced Arthritic Mice

doi: 10.1155/2015/765490

Figure Lengend Snippet: Construction of the DNA vaccine. (a) The maps of recombinant DKK1 DNA vaccine. (b) The amino acid sequence of human DKK1. (c) The recombinant amino acid sequence of DKK1 DNA vaccine. Red line, 110–144aa; green line, 153–181aa; orange line, 182–216aa; blue line, 228–253aa; black line, signal peptide; box, PADRE.

Article Snippet: After blocking with 5% horse serum, the sections were incubated with a goat anti-human DKK1 antibody (5 μ g/mL, R&D Systems) overnight at 4°C.

Techniques: Recombinant, Sequencing

Journal: BioMed Research International

Article Title: Designation of a Novel DKK1 Multiepitope DNA Vaccine and Inhibition of Bone Loss in Collagen-Induced Arthritic Mice

doi: 10.1155/2015/765490

Figure Lengend Snippet: Expression and immunogenicity of the DNA vaccine. (a–c) Expression of the multiepitope protein in vitro and in vivo were determined in cell culture supernatants by ELISA, cell lysates by Western blotting, and the muscles of mice by immunohistochemical analysis. (d-e) The DNA vaccine induced a specific IgG antibody against human DKK1. The titer and the end-point titer of the specific antibody were tested by ELISA. Bars indicate 100 μ m. Data are expressed as the mean ± SEM, * P < 0.05, *** P < 0.001.

Article Snippet: After blocking with 5% horse serum, the sections were incubated with a goat anti-human DKK1 antibody (5 μ g/mL, R&D Systems) overnight at 4°C.

Techniques: Expressing, Immunopeptidomics, In Vitro, In Vivo, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Muscles, Immunohistochemical staining