Journal: Virology Journal
Article Title: Translation of nervous necrosis virus involves eIF4E but not RPS6 phosphorylation and viral particle assembly in remodeled microtubule-organizing center
doi: 10.1186/s12985-025-02799-3
Figure Lengend Snippet: Inhibition of MNK1 phosphorylation in GGNNV-infected GB cells reduces p-MNK1 and p-eIF4E production as well as coat protein translation. A GB cells were infected with GGNNV (MOI = 100). Cells were fixed at 0, 12 and 24 hpi and immunostained for p-MNK1 (green) and coat protein (red). B The cytotoxic effect of MNK1 phosphorylation inhibitor, CGP57380 on GB cells was evaluated by MTT assay. GB cells cultured in a 96-well plate were treated with different concentrations of CGP57380 for 24 h. The cell viabilities were detected with a MTT kit. Values are presented as mean ± SD ( n = 3). C Immunocytochemical staining of p-MNK1 (green) and coat protein (red) in DMSO treated (control) GGNNV-infected GB cells. D Immunocytochemical staining of p-MNK1 (green) and coat protein (red) in 10 µM CGP57380 treated GGNNV-infected GB cells. E Relative coat protein expressions (%) (co-stained with p-MNK1) in DMSO and CGP57380 treated cells were analyzed at 6, 12, 18 and 24 hpi ( n = 54 to 66 cells). F Relative fluorescence intensity of p-MNK1 in DMSO and CGP57380 treated GGNNV-infected cells ( n = 50 to 60 cells). G Relative fluorescence intensity of p-eIF4E in DMSO and CGP57380 treated GGNNV-infected cells ( n = 50 to 60 cells). DMSO and inhibitor treated GB cells were infected with GGNNV (MOI = 100). The samples were collected and fixed at 0, 6, 12, 18 and 24 hpi and then proceeded with immunocytochemical staining using anti-p-eIF4E and anti-RG-M18 antibodies (image is shown in supplementary Fig. ). The mean ± SD for coat protein, p-MNK1 and p-eIF4E were plotted. ns, not significant; **, P < 0.002; ****, P < 0.0001 (two-way ANOVA test). The nuclei (blue) were stained with DAPI. Scale bar = 20 μm. DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; GB, grouper brain; GGNNV, giant grouper nervous necrosis virus; hpi, hour post infection; MOI, multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SD, standard deviation
Article Snippet: Rabbit anti-phospho-eIF4E (p-eIF4E) (Ser209) polyclonal antibody (#9741), rabbit anti-phospho-eIF4E-BP1 (p-eIF4E-BP) (Ser65) monoclonal antibody (D9G1Q) (#13443), rabbit anti-phospho-MNK1 (p-MNK1) (Thr197/202) polyclonal antibody (#2111), rabbit anti-phospho-p44/42 MAPK (p-ERK) (Thr202/204) monoclonal antibody (D13.14.4E) (#4370), rabbit anti-phospho-p38 MAPK (p-p38) (Thr180/Tyr182) monoclonal antibody (D3F9) (#4511), rabbit anti-phospho-S6 ribosomal protein (p-RPS6) (Ser235/236) monoclonal antibody (D57.2.2E) (#4858) and rabbit anti-phospho-p70S6 kinase (p-p70S6K) (Thr389) monoclonal antibody (#9205) were purchased from Cell Signaling Technology.
Techniques: Inhibition, Phospho-proteomics, Infection, MTT Assay, Cell Culture, Staining, Control, Fluorescence, Virus, Standard Deviation
