Journal: Veterinary research

Article Title: PGK1 enhances productive bovine herpesvirus 1 infection by stimulating β-catenin-dependent transcription.

doi: 10.1186/s13567-025-01480-5

Figure Lengend Snippet: Figure 4 PGK1 plays an essential role in BoHV-1 productive infection. A MDBK cells in 6-well plates were transfected with either scrambled siRNA (200 pmol) or three individual siRNA targeting PGK1 (200 pmol), referred to as siRNAPGK1-1, siRNAPGK1-2, and siRNAPGK1-3, respectively. At 48 h post-transfection, PGK1 protein levels were detected via western blot. B, C MDBK cells in 6-well plates were transfected with 200 pmol of scrambled siRNA, siRNAPGK1-1, and siRNAPGK1-2, respectively. After transfection for 36 h, the cells were infected with BoHV-1 (MOI = 0.1) for one h. After three washes with PBS, the fresh medium was replaced. At 12 and 24 hpi virus yield in the cell cultures was measured, with results expressed as TCID50/mL (B), and levels of viral DNA were examined from intracellular content using qPCR with gC-specific primers (C). D The cytotoxicity of NG52 (5μM) in MDBK cells for 24 h was analysed by Trypan-blue exclusion test. E and F MDBK cells in 24-well plates pretreated with either DMSO control or NG52 at indicated concentrations were infected with BoHV-1 (MOI = 0.1) for one hour along with treatment-indicated chemicals. After three washing with PBS, fresh media containing DMSO control or NG52 were added for further incubation. At 12 and 24 hpi, the virus titers were measured, with the results expressed as TCID50/mL (E), and the intracellular content of virus genomic DNA was determined using relative qPCR with gC-specific primers (F). G and H MDBK cells in 6-well plates were transfected with 200 pmol of scrambled siRNA or siRNAPGK1-1. After transfection for 36 h, they were infected with BoHV-1 (MOI = 0.1) for 24 h. Total RNA was purified, and mRNA levels of bICP27 (G) and VP16 (H) were subsequently detected via qRT-PCR, respectively. The results shown are the means of three independent experiments, with error bars indicating standard deviations. Significance was assessed with Student’s t-test (* p < 0.05; ns = insignificant).

Article Snippet: The cells were treated with the chemical NG52 (MCE, cat# HY-15154) at a specified concentration for 1 h at 37 °C.

Techniques: Infection, Transfection, Western Blot, Virus, Control, Incubation, Purification, Quantitative RT-PCR

Journal: Veterinary research

Article Title: PGK1 enhances productive bovine herpesvirus 1 infection by stimulating β-catenin-dependent transcription.

doi: 10.1186/s13567-025-01480-5

Figure Lengend Snippet: Figure 6 PGK1 positively regulates β-catenin expression. A MDBK cells in 6-well plates were treated with either DMSO control or NG52 at indicated concentrations for 24 h. The cell lysates were prepared and subjected to western blot to detect β-catenin protein levels. C MDBK cells in 6-well plates were transfected with 200 pmol of scrambled siRNA, siRNAPGK1-1, siRNAPGK1-2, and siRNAPGK1-3, respectively. At 48 h after transfection, β-catenin protein levels were detected by western blot. E PGK1 plasmid along with empty vector at the indicated dose were transfected into Neuro-2A cells in 6-well plates using lipofectamine 3000; after transfection for 48 h, the cells were either collected for the detection of β-catenin protein levels via western blot. G MDBK cells in 6-well plates were transfected with 200 pmol of scrambled siRNA or siRNAPGK1-1, siRNAPGK1-2, and siRNAPGK1-3, respectively. At 36 h after transfection, the cells were infected with BoHV-1 at an MOI of 0.1 for 24 h. Cell lysates were prepared with RIPA buffer and then protein levels of β-catenin were analysed using western blotting. GAPDH was probed as a loading control. B, D, F, and H Band intensity was analysed using the Image J software. The control was arbitrarily set as either 1 or 100%. The results shown are representations of three independent experiments, with error bars indicating standard deviations. Significance was assessed with Student’s t-test (*p < 0.05; ns = not significant).

Article Snippet: The cells were treated with the chemical NG52 (MCE, cat# HY-15154) at a specified concentration for 1 h at 37 °C.

Techniques: Expressing, Control, Western Blot, Transfection, Plasmid Preparation, Infection, Software

Journal: Veterinary research

Article Title: PGK1 enhances productive bovine herpesvirus 1 infection by stimulating β-catenin-dependent transcription.

doi: 10.1186/s13567-025-01480-5

Figure Lengend Snippet: Figure 7 PGK1 stimulates β-catenin-dependent transcription. A MDBK cells in 60 mm dishes were mock infected or infected with BoHV-1(MOI = 0.1) for 24 h. Cell lysates were subjected to IP using antibodies against either PGK1 β-catenin or isotype IgG. Then both β-catenin and PGK1 were detected by western blot. The data shown are representative of three independent experiments. B Neuro-2A cells were co-transfected with 0.1 μg of the Super 8 × TOPFlash luciferase reporter construct, 0.01 μg of the Renilla reporter construct, and 0.25 μg of β-cateninS33Y mutant (S33Y) plasmid, together with increasing concentrations of a plasmid expressing PGK1 (0.5 or 1 μg) to examine the effect that PGK1 has on TCF promoter activity. At 48 h after transfection, dual luciferase assays were performed. C MDBK cells in 12-well plates (60% confluent) were transfected with 0.4 μg of the Super 8 × TOPFlash luciferase reporter construct and 0.05 μg of the Renilla reporter construct that was used as an internal control to allow normalisation of promoter activity. After transfection for 36 h, the cells were infected with BoHV-1 (MOI = 1) for 24 h along with treatment of either DMSO control or NG52 at the designated concentrations. Dual luciferase assays were performed 24 h after infection. The results shown are the average of three independent experiments, with error bars indicating standard deviations. Significance was assessed with Student’s t-test (*p < 0.05; ns, not significant).

Article Snippet: The cells were treated with the chemical NG52 (MCE, cat# HY-15154) at a specified concentration for 1 h at 37 °C.

Techniques: Infection, Western Blot, Transfection, Luciferase, Construct, Mutagenesis, Plasmid Preparation, Expressing, Activity Assay, Control

Journal: Journal of molecular and cellular cardiology

Article Title: Stem Cell Factor is Responsible for the Rapid Response in Mature Mast Cell Density in the Acutely Stressed Heart

doi: 10.1016/j.yjmcc.2012.07.011

Figure Lengend Snippet: Stem cell factor (SCF) concentration in left ventricular (LV) tissue slices (3A) and the conditioned medium (3B) and mature mast cell (MC) density (3C) that were incubated with media alone (control), compound 48/80 (20 μg/ml), or 48/80 plus chymostatin for 16 hrs. SCF levels in LV tissue slices and culture media were normalized with total protein and tissue slice weight, respectively, and are presented as mean ± SEM. Number within bars represents the number of rat hearts in the group. * p < 0.01 vs. control and chymostatin groups.

Article Snippet: To determine the effects of the MC degranulation on SCF levels, LV slices from each heart were randomly separated into three groups with five or more slices per well: 1) culture media alone; 2) culture media with the MC secretagogue, compound 48/80 (20 μg/ml; Sigma, St Louis, MO); 3) culture media with compound 48/80 and anti-SCF; and 4) culture media with compound 48/80 and chymostatin (10 μM, MP Biomedicals, Solon, OH).

Techniques: Concentration Assay, Incubation

Journal: Frontiers in Immunology

Article Title: Annexin A1 Mimetic Peptide Ac 2-26 Modulates the Function of Murine Colonic and Human Mast Cells

doi: 10.3389/fimmu.2021.689484

Figure Lengend Snippet: Cytokine production by colon explants from C57BL/6 WT and C57BL/6 IL-4 KO mice treated in vivo with 1 mg/kg i.p. Ac 2-26 (+) or PBS (-). ELISA determined the concentration of IL-12 (A) , TNF-α (B) , IL-10 (C) and IL-4 (D) in explants stimulated for 24 h, with 20µg/mL compound 48/80 and with or without 3µM Ac2-26 treatment. Experiments were performed twice with n = 4 animals/group. Values of concentration in pg/mL are expressed as the mean ± S.D, n = 8. Data were analyzed using one-way ANOVA (post Tukey multiple-comparison test). **p <0.01 vs. respective WT group. ## p <0.01 vs. untreated in vivo and ex vivo (-/-) and && p <0.01 vs treated in vivo and not treated ex vivo (-/+).

Article Snippet: Remarkably, our study demonstrated that the AnxA1-derived peptide Ac 2-26 inhibited in vitro the degranulation of HMC-1 cells stimulated with compound 48/80 in FPR-dependent manner, as assessed by blocking the degranulation system using the antagonist BOC-2.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Concentration Assay, Ex Vivo

Journal: Frontiers in Immunology

Article Title: Annexin A1 Mimetic Peptide Ac 2-26 Modulates the Function of Murine Colonic and Human Mast Cells

doi: 10.3389/fimmu.2021.689484

Figure Lengend Snippet: Effect of peptide Ac2-26 on MC degranulation in colon explants. C57BL/6 WT and C57BL/6 interleukin-4- KO mice were pre-treated with PBS (-) or 3µM Ac2 -26 (+) and stimulated with 20µg/mL compound 48/80 ex vivo for 24 (h) (A) Representative colon sections with examples of intact (arrowheads) and degranulated (arrows) MCs. Staining: Toluidine Blue. Sections: 4μm. Scale bars: 20 μm. (B) Degranulation of MCs was assessed by an enzymatic assay. Experiments were performed twice with n = 4 animals/group. Values of the percentage of β hexosaminidase release are expressed as the mean ± S.D., n = 8. Data were analyzed using one-way ANOVA (post Tukey multiple-comparison test). **p <0.01 compared to respective WT group. ## p <0.01 compared to untreated in vivo and ex vivo (-/-) and && p <0.01 compared to treated and not treated ex vivo (-/+).

Article Snippet: Remarkably, our study demonstrated that the AnxA1-derived peptide Ac 2-26 inhibited in vitro the degranulation of HMC-1 cells stimulated with compound 48/80 in FPR-dependent manner, as assessed by blocking the degranulation system using the antagonist BOC-2.

Techniques: Ex Vivo, Staining, Enzymatic Assay, In Vivo

Journal: Frontiers in Immunology

Article Title: Annexin A1 Mimetic Peptide Ac 2-26 Modulates the Function of Murine Colonic and Human Mast Cells

doi: 10.3389/fimmu.2021.689484

Figure Lengend Snippet: Exogenous AnxA1 (peptide Ac2-26) inhibits HMC-1 cell line degranulation induced by compound 48/80. HMC-1 cells were pre-treated with 3, 5, or 10µM peptide Ac2-26 for 30 min in the absence or presence of FPRs antagonist BOC-2 (5µg/mL). Degranulation was assessed measuring the activity of β-hexosaminidase in culture supernatants. Results are expressed as percentage of enzyme release. All experiments were conducted in triplicate. Data are expressed as the mean ± S.D from four different experiments and analyzed using one-way ANOVA. ***p <0.001 compared to cells treated with 48/80 alone or 48/80 + Ac2-26 (3 or 5 µM); **p <0.01 compared to cells treated with 48/80 + Ac2-26 (10 µM) + BOC-2; ## p <0.01 compared to cells treated with 48/80 alone.

Article Snippet: Remarkably, our study demonstrated that the AnxA1-derived peptide Ac 2-26 inhibited in vitro the degranulation of HMC-1 cells stimulated with compound 48/80 in FPR-dependent manner, as assessed by blocking the degranulation system using the antagonist BOC-2.

Techniques: Activity Assay

Journal: Frontiers in Immunology

Article Title: Annexin A1 Mimetic Peptide Ac 2-26 Modulates the Function of Murine Colonic and Human Mast Cells

doi: 10.3389/fimmu.2021.689484

Figure Lengend Snippet: Exogenous AnxA1 (peptide Ac2-26) did not alter viability of HMC-1 cell line. Cells were pre-treated with 3, 5, or 10µM peptide Ac2-26 for 30 min in the absence or presence of FPRs antagonist BOC-2 (5µg/mL). The cells were then stimulated with 20 µg/mL compound 48/80 for 30 min. Cell viability was assessed by trypan blue exclusion assay and results are expressed as percentage. All experiments were performed in triplicate. Data are expressed as the mean ± S.D from four different experiments.

Article Snippet: Remarkably, our study demonstrated that the AnxA1-derived peptide Ac 2-26 inhibited in vitro the degranulation of HMC-1 cells stimulated with compound 48/80 in FPR-dependent manner, as assessed by blocking the degranulation system using the antagonist BOC-2.

Techniques: Trypan Blue Exclusion Assay