The impact of 12C6 heavy ion irradiation-induced cellular mutations on the replication of the foot-and-mouth disease virus and the role of Cbr3

Figures

• 

  Fig. 1. A BHK-21 cells were induced with 12C6 heavy ions at doses of 5 Gy, 10 Gy, and 15 Gy. Cell viability was assessed via trypan blue staining and detected via a Count STAR (Countstar Rigel S2, RY074B2001), and the mortality rate was calculated. The data represent the means and standard errors from six independent experiments. B Changes in cell density over time (0 to 72 h) were monitored for control (BHK-21) cells and mutant BHK-7 and BHK-5 cells via a Count STAR. All the cells to be detected had the same initial density. Growth curves were plotted on the basis of the mean ± standard error from three independent experiments. C Fourteen mutant cell lines and control (BHK-21) cells were adjusted to the same initial density and infected with FMDV at an MOI of 1.0 for 16 h. The content of FMDV antigen 146 s was quantified. The results are presented as the means and standard errors from three independent experiments. D The expression level of the FMDV 3D gene in 14 mutant cell lines and control BHK-21 cells infected with FMDV (MOI = 1.0) for 16 h was measured by qRT-PCR with GAPDH as the reference. Relative expression levels were calculated via the 2−ΔΔCt method. The data represent the…

• 

  Fig. 2. A Control BHK-21 cells and mutant BHK-7 and BHK-5 cells were passaged 9 times and infected with FMDV (MOI = 1.0) for 16 h every 3 passages to monitor changes in the content of the FMDV antigen 146S. B Control BHK-21 cells and mutant BHK-7 and BHK-5 cells were passaged 9 times and infected with FMDV (MOI = 1.0) for 16 h every 3 passages to assess changes in TCID50 values. The data represent the means ± standard deviations from three independent measurements. The data are presented as the means ± SDs from three independent experiments and were analyzed via Student's two-tailed unpaired t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

• 

  Fig. 3. A Statistical graph of significantly different proteins between BHK-5, BHK-7, and the control (BHK-21); B Statistical graph of significantly different proteins after FMDV infection in BHK-5, BHK-7, and the control (BHK-21); C and D Heatmaps of significantly different protein expression before and after FMDV infection in BHK-5, BHK-7, and the control (BHK-21)

• 

  Fig. 4. A KEGG pathway map associated with proteins differentially expressed between BHK-5 and BHK-21 cells. B KEGG pathway map associated with proteins differentially expressed between BHK-7 and BHK-21 cells. C KEGG pathway map associated with proteins differentially expressed between BHK-5 and BHK-21 cells post-FMDV infection. D KEGG pathway map associated with proteins differentially expressed between BHK-7 and BHK-21 cells post-FMDV infection. The x-axis represents the -log10(p-value) derived from Fisher's exact test, indicating the statistical significance of pathway enrichment. The y-axis lists the names of the pathways. The upregulated and downregulated pathways are denoted by red (right) and blue (left) bars, respectively

• 

  Fig. 5. The multi-group differential volcano plots generated on the basis of the log fold change (logFC) and p-value of differentially expressed proteins (DEPs) in BHK-5, BHK-7, and control BHK-21 cells. The top 10 proteins from each group are labeled. The red dots indicate upregulated DEPs, the green dots denote downregulated DEGs, and the gray dots represent genes whose expression did not significantly change

• 

  Fig. 6. A Cbr3 expression was detected via Western blotting via an anti-Cbr3 antibody, with β-actin serving as the loading control to ensure uniform sample loading. B WT-BHK-21 and BHK-21-KO-Cbr3 cells were infected with FMDV (MOI = 1.0). At 16 h postinfection, the protein levels of FMDV VP1 were assessed via Western blotting, with β-actin used as the internal reference. C WT-BHK-21 and BHK-21-KO-Cbr3 cells were seeded in T25 flasks at a density of 2.0 × 103 cells/mL. Trypsinized cells were counted every 4 h to construct growth curves. D: WT-BHK-21 and BHK-21-KO-Cbr3 cells were seeded at a density of 3.0 × 105 cells/mL and infected with FMDV (MOI = 1.0) for 16 h. The relative expression of FMDV 3D mRNA was quantified via qRT-PCR. All experiments were performed in triplicate, and the data are presented as the means ± SDs (n = 3). E: WT-BHK-21 and BHK-21-KO-Cbr3 cells were infected with FMDV (MOI = 1.0) at a density of 3.0 × 105 cells/mL for 16 h. The content of FMDV antigen 146 s was measured. The data are presented as the means ± SDs from three independent experiments and were analyzed via Student's two-tailed unpaired t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

• 

  Fig. 7. A WT-BHK-21 and BHK-21-KO-Cbr3 cells were infected with FMDV (MOI = 1.0). At 4, 8, and 16 h postinfection, the protein levels of FMDV VP1 were assessed via Western blotting, with β-actin used as the internal reference. B: TCID₅₀ values were determined by infecting WT-BHK-21 and BHK-21-KO-Cbr3 cells with FMDV. The experiments were conducted three times, and the data are expressed as the means ± SDs (n = 3). C: WT-BHK-21 and BHK-21-KO-Cbr3 cells were infected with FMDV (MOI = 1.0) for 8 h. Immunofluorescence staining followed by confocal microscopy was used to observe the signal of the FMDV protein VP1. The green signal represents VP1, the nucleus was stained with Hoechst 33,258, and the red signal represents β-actin. The data are presented as the means ± SDs from three independent experiments and were analyzed via Student's two-tailed unpaired t-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

• 

  Fig. 8. A represents the synthesis pathway of prostaglandin E2 (PGE2) in cells (AA arachidonic acid; PGH2 prostaglandin H2; PGE2 prostaglandin E2; PGF2α prostaglandin F2α). B PGE2 content in cells was determined via a PGE2 ELISA kit. The experiment included three replicates, and the results are expressed as the mean ± standard deviation (SD) (n = 3). The protein expression level of Cbr3 in BHK-21-ko-Cbr3 and control BHK-21 cells was evaluated. D. Wild-type BHK-21 cells were cultured in 6-well plates and treated with 0.005, 0.01, or 0.02 nmol/ml PGE2. The cells were infected with the foot-and-mouth disease virus (FMDV) for 16 h at a multiplicity of infection (MOI) of 1.0. The relative expression level of FMDV 3D mRNA was detected via real-time quantitative PCR (qRT-PCR). The experiment included three replicates, and the results are expressed as the means ± SDs (n = 3). When exogenous PGE2 was added to BHK-5 cells, the protein expression of Cbr3 was significantly greater than that in control BHK-21 cells. The cells were infected with FMDV (MOI = 1.0) for 16 h. The expression of the FMDV VP1 protein was detected by Western blotting, with β-actin used as the loading control. F. BHK-5 cells…