pe stain buffer (fbs  (Becton Dickinson)

Journal: Veterinary Research

Article Title: Neu1 inhibition restrains BCoV replication and modulates ZBP1-dependent PANoptosis

doi: 10.1186/s13567-026-01729-7

Figure Lengend Snippet: BCoV infection induces the expression of PANoptosis-related genes in MDBK cells. A Western blot analysis of RIPK3, RIPK1, p-MLKL, and ZBP1 protein levels. B , C Relative mRNA levels of ZBP1 ( B ) and RIPK3 ( C ) determined by qPCR. D , E Protein ( D ) and mRNA ( E ) expression of CASP8. F Flow cytometric analysis of apoptosis via Annexin V-PE/7-AAD staining in control and BCoV-infected cells. G , H Protein ( G ) and mRNA ( H ) expression of GSDMD, CASP1, and ASC. I Relative mRNA expression of IL-1β . J , K Protein ( J ) and mRNA ( K ) expression of Neu1. L Cellular sialic acid levels, which decreased in a dose-dependent manner upon BCoV infection. Quantitative densitometry of the western blot results (normalized to β-actin/GAPDH) is shown in Additional file . The data are presented as the means ± SDs ( n = 3). Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The pellets were washed three times with 1× PBS and subsequently resuspended in 1× Annexin V-PE binding buffer (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCl 2 solution; Servicebio, Wuhan) at the appropriate cell density.

Techniques: Infection, Expressing, Western Blot, Staining, Control

Journal: Veterinary Research

Article Title: Neu1 inhibition restrains BCoV replication and modulates ZBP1-dependent PANoptosis

doi: 10.1186/s13567-026-01729-7

Figure Lengend Snippet: The interaction between Neu1 and ZBP1 regulates apoptosis and sialic acid levels in BCoV-infected MDBK cells. A , B Schematic representation and validation of stable MDBK Neu1kd and MDBK ZBP1kd cell lines. C Apoptosis analysis of WT, MDBK Neu1kd , and MDBK ZBP1kd cells following BCoV infection via Annexin V-PE/7-AAD flow cytometry. D , E Viral titers determined via the Reed–Muench method ( D ) and relative BCoV mRNA expression levels ( E ). F Immunofluorescence localization of Neu1 (red) and ZBP1 (green) in WT, MDBK Neu1kd , and MDBK ZBP1kd cells. Nuclei were stained with DAPI (blue), and merged signals (yellow) indicate colocalization. G Cellular sialic acid levels significantly decreased in both MDBK Neu1kd and MDBK ZBP1kd cells. H Immunofluorescence analysis further demonstrated that Neu1 or ZBP1 knockdown markedly reduced the fluorescence signal intensity during BCoV infection. The data are presented as the means ± SDs ( n = 3). The quantification of fluorescence intensity is shown in Additional file . Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The pellets were washed three times with 1× PBS and subsequently resuspended in 1× Annexin V-PE binding buffer (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCl 2 solution; Servicebio, Wuhan) at the appropriate cell density.

Techniques: Infection, Biomarker Discovery, Flow Cytometry, Endpoint Dilution Assay, Expressing, Immunofluorescence, Staining, Knockdown, Fluorescence