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