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
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Cell Signaling Technology Inc manufactures this product 
![DHA accelerates caspase-1 protein synthesis in a p38MAP-dependent manner (A) Protein synthesis signaling diagram. Inhibitors for signaling components are denoted in red. (B) ES2 and SK-OV3 cells were treated with DHA at IC50 for 16 or 24 h, followed by western blotting to detect phosphor-p38MAPK, p38MAPK, phosphor-Akt, Akt, phosphor-Mnk1, Mnk1, phosphor-Erk, Erk, <t>phosphor-eIF4E,</t> and eIF4E with the respective antibodies. (C) ES2 cells were treated with DHA in the absence or presence of AZD5383, Trametinib, BIRB796, or Rapamycin for 24 h, followed by western blotting to detect caspase-1 and GAPDH with the respective antibodies. (D) ES2 and SK-OV3 cells were cultured in a methionine-free medium for 2 h, and then fed with a medium containing AHA in the absence or presence of BIRB796 for 16 h. Cells were lysed and AHA-incorporated proteins were isolated for western blotting to detect caspase-1 with caspase-1 antibody. Cell lysates before isolating AHA-incorporated proteins were also subjected to western blotting to detect GAPDH to ensure equal loading. (E) ES2, SK-OV3, and OCC1 cells were treated with vehicle or 100 μM DHA in the absence or presence of AZD5363, Trametinib, Rapamycin, BIRB796, or 796 CPG57380 for 24 h followed by MTT assay to analyze cell viability. % of cell viability was calculated as [(Control – Treatment)/Control] x 100. Control: vehicle-treated; Treatment: DHA or DHA + inhibitors. Data are means ± SEM. ∗∗∗, p < 0.001.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6124/pmc10696124/pmc10696124__gr8.jpg)
