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: 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 TCID 50 /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 TCID 50 /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: 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: 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: Advanced Science

Article Title: Targeting Fibrotic Scarring by Mechanoregulation of Il11ra1 + /Itga11 + Fibroblast Patterning Promotes Axon Growth after Spinal Cord Injury

doi: 10.1002/advs.202513476

Figure Lengend Snippet: MMP7 was a key factor in regulating the response of fibroblasts to environmental mechanical strength. A) KEGG functional enrichment analysis of signaling pathways in fibroblast clusters based on scRNA‐seq. B) Venn diagram illustrated the relationships of the DEGs between the SCI and Medium groups, High and Medium groups, and Low and Medium groups from the Bulk RNA sequencing (bulk RNA‐seq). C) Venn diagram illustrated the overlapped DEG between the 23 DEGs in bulk RNA‐seq and 126 downstream effector molecules of the Wnt signaling pathway. The Heatmap showed the expression of Mmp7 gene among samples based on bulk RNA‐seq. D) The representative chopped Western blot images and quantitation of the expression of MMP7 in vivo and in vitro, respectively. N = 3 samples, respectively. E) Schematic of the experimental design to investigate the role of MMP7 in vivo and in vitro. F) Representative multichannel immunostaining images showed the expression of Col1a1, Itga11, and Il11ral in the longitudinal sections of the spinal cord at 2 wpi following the intervention with MMP7 on Medium hydrogel group. The separate channels were presented in the right rows. G)The quantifications of the proportion of Itga11 + /Il11ra1 + cells within the Col1a1 + population following the intervention with MMP7 on Medium hydrogel group. N = 6 animals. H) Representative multichannel immunostaining images showed the expression of Col1a1, Itga11, and Il11ral in the longitudinal sections of the spinal cord at 2 wpi following the intervention with MMP‐7‐IN‐1 on SCI, High hydrogel and Low hydrogel group, respectively. The separate channels were presented in the right rows. I) The quantifications of the proportion of Itga11 + /Il11ra1 + cells within the Col1a1 + population following the intervention with MMP‐7‐IN‐1 on SCI, High hydrogel and Low hydrogel group. N = 6 animals. J) Representative multichannel immunostaining images revealed the expression of Col1a1 in the longitudinal sections of the spinal cord at 2 wpi following the intervention with MMP7 on Medium hydrogel group. The blue boxed regions were the higher magnifications of the color‐coded images revealing the orientation of Col1a1 using OrientationJ. The Color scale bars represented Col1a1 orientation. K,L) The quantifications of the orientations of Col1a1 following the intervention with MMP7 on Medium hydrogel group. M) Representative multichannel immunostaining images revealed the expression of Col1a1 in the longitudinal sections of the spinal cord at 2 wpi following the intervention with MMP‐7‐IN‐1 on SCI, High hydrogel and Low hydrogel group, respectively. The blue boxed regions were the higher magnifications of the color‐coded images revealing the orientation of Col1a1 using OrientationJ. The Color scale bars represented Col1a1 orientation. N,O) The quantifications of the orientations of Col1a1 following the intervention with MMP‐7‐IN‐1 on SCI, High hydrogel and Low hydrogel group, respectively. P) The representative images showing the co‐expression of DetyrTub and phalloidin in the fibroblasts that migrated on the hydrogel side (right side) following the intervention with MMP7 on Medium hydrogel group. Q) The quantification of the relative fluorescence intensity of DetyrTub following the intervention with MMP7 on Medium hydrogel group. N = 20 cells. R) The representative images showing the co‐expression of DetyrTub and phalloidin in the fibroblasts that migrated on the hydrogel side (right side) following the intervention with MMP‐7‐IN‐1 on High hydrogel group. S) The quantification of the relative fluorescence intensity of DetyrTub following the intervention with MMP‐7‐IN‐1 on High hydrogel group and Low hydrogel group, respectively. N = 20 cells. T) The representative images showing the co‐expression of vinculin and phalloidin in the fibroblasts that migrated on the hydrogel side (right side) following the intervention with MMP7 on Medium hydrogel group. U) The quantification of the relative fluorescence intensity of the vinculin with longitudinal/lateral ratio following the intervention with MMP7 on Medium hydrogel group. N = 9 cells. V) The representative images showing the co‐expression of vinculin and phalloidin in the fibroblasts that migrated on the hydrogel side (right side) following the intervention with MMP‐7‐IN‐1 on High hydrogel group. W) The quantification of the relative fluorescence intensity of the vinculin with longitudinal/lateral ratio following the intervention with MMP‐7‐IN‐1 on High hydrogel group and Low hydrogel group, respectively. N = 9 cells, respectively. Data were shown as the mean ± SD; p values were determined by two‐tailed one‐way ANOVA with Tukey's multiple‐comparisons test or two‐tailed paired t ‐tests.

Article Snippet: For in vitro experiments, MMP7 protein (100 μM, HY‐P700574, MedChem‐Express, USA) or its small molecule inhibitor MMP‐7‐IN‐1 (1 μM, HY‐151540, MedChem‐Express, USA) was added to the culture medium.

Techniques: Functional Assay, Protein-Protein interactions, RNA Sequencing, Expressing, Western Blot, Quantitation Assay, In Vivo, In Vitro, Immunostaining, Fluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting Fibrotic Scarring by Mechanoregulation of Il11ra1 + /Itga11 + Fibroblast Patterning Promotes Axon Growth after Spinal Cord Injury

doi: 10.1002/advs.202513476

Figure Lengend Snippet: Mechanotransduction induced the Itga11 + /Il11ra1 + fibroblast subset via LRP6‐Wnt/β‐Catenin‐MMP7 signaling axis. A–C) Representative chopped Western blot images A) and quantitation of the expression of phosphorylated β‐catenin B) and β‐catenin C) in vivo. N = 3 samples, respectively. D–F) Representative chopped Western blot images D) and quantitation of the expression of phosphorylated β‐catenin E), and β‐catenin F) in vitro. N = 3 samples, respectively. G) Representative multichannel immunostaining images revealing the expression active β‐catenin and phalloidin in the migrating fibroblasts in different groups. The white boxed regions were the high magnifications of the active β‐catenin signal and DAPI, which were presented in separate channels. H)The quantifications of the percentage of active β‐catenin entering nucleus. N = 9 cells. I) Heatmap showing the expression levels of LRP5 and LRP6 from scRNA‐seq. J,K) The representative chopped Western blot images J) and quantitation K) showing the expression of LRP6 and phosphorylated LRP6 proteins in vivo. N = 3 animals. L) Schematic of the experimental design for the in vitro validation. M) Western blot results of related proteins expressions in vitro following intervention with HLY78 and Salinomycin respectively. N) Representative multichannel immunostaining images showing the expression active β‐catenin and phalloidin in the migrating fibroblasts in Medium hydrogel group following intervention with HLY78. The white boxed regions were higher magnifications of the active β‐catenin signal and DAPI, which were presented in separate channels. The quantifications of the proportion of active β‐catenin entering nucleus following the intervention with HLY78 were listed on the right. N = 9 cells. O,P) Representative multichannel immunostaining images showing the expression active β‐catenin and phalloidin in the migrating fibroblasts in High hydrogel group O) and Low hydrogel group P) following intervention with Salinomycin. The white boxed regions were higher magnifications of the active β‐catenin signal and DAPI, which were presented in separate channels. The quantifications of the proportion of active β‐catenin entering nucleus following the intervention with HLY78 were listed on the right. N = 9 cells, respectively. Q) Flow cytometric analysis of Itgall + Il11ra1 + fibroblasts in vitro in Medium hydrogel group following intervention with HLY78. N = 3 samples. R,S) Flow cytometric analysis of Itgall + Il11ra1 + fibroblasts in vitro in High hydrogel group R) and Low hydrogel group S) following intervention with Salinomycin. N = 3 samples, respectively. T) Representative chopped Western blot images showing the expression of the related proteins in vivo following intervention with HLY78 and Salinomycin respectively. U) Schematic representation of the LRP6‐Wnt/β‐Catenin‐MMP7 pathway axis. Data were shown as the mean ± SD; p values were determined by two‐tailed one‐way ANOVA with Tukey's multiple‐comparisons test or two‐tailed paired t ‐tests.

Article Snippet: For in vitro experiments, MMP7 protein (100 μM, HY‐P700574, MedChem‐Express, USA) or its small molecule inhibitor MMP‐7‐IN‐1 (1 μM, HY‐151540, MedChem‐Express, USA) was added to the culture medium.

Techniques: Western Blot, Quantitation Assay, Expressing, In Vivo, In Vitro, Immunostaining, Biomarker Discovery, Two Tailed Test