Fig 2: The effect of eIF4E overexpression on social cognitive behavior in mice was assessed. A, In the first phase of the three-box social experiment, the duration of contact time between control and eIF4Eki/ki mice and Stranger Mouse 1 was measured. B, In the second phase of the three-box social experiment, the duration of contact between control and eIF4Eki/ki mice and Stranger Mouse 2 was recorded. C, D, The objective of the self-care experiments was to quantify the time spent on self-care activities and the number of self-care procedures performed by control and eIF4Eki/ki mice. E–G, Open-field assays were conducted to assess the total distance traversed, duration of open-field stays, and number of open-field stays by control and eIF4Eki/ki mice. H, The trajectories of mice in open-field experiments are plotted. I–M, The water maze tests included a 5 d localization navigation phase, during which platform latency and destination quadrant residence time were measured in control and eIF4Ekki/ki mice. On the sixth day of the experiment, the time spent by the subjects on the platform and the number of visits to the destination quadrant were recorded. N, The trajectories of mice in the Morris water maze test are plotted. The Mann–Whitney U test was used to statistically analyze data from the three-chamber social interaction test, open-field test, and self-grooming test, along with the duration spent and the frequency of entries into the target quadrant during the water maze test. Escape latency during the learning phase of the water maze was analyzed using two-way ANOVA followed by Tukey's multiple-comparisons test. Data are presented as mean ± standard deviation, and statistical significance is denoted as *p < 0.05, **p < 0.01, and ***p < 0.0001 (control, n = 12; eIF4Eki/ki, n = 12).

Fig 3: The effect of cap-proximal nucleotides on eIF4E binding affinity and activity.(A) The relative level of eIF4E reads (mean of two independent replicates) in the indicated fractions in basal (cont) and GS conditions. (B) The relative mRNA levels (mean of two independent replicates) of eIF4E in basal (cont) and GS conditions. (C) Representative immunoblot of total lysate using eIF4E and Tubulin antibodies following GS of MEFs for the indicated times. (D) Upper panel – the change in the intrinsic fluorescence of eIF4E (300 nM) in response to increasing concentrations of capped RNA ligands (1.25 nM–5 μM) or cap analog (1.25 nM–10 μM). The graphs represent the mean of three independent experiments with two different protein preparations. The bottom panel shows the calculated dissociation constant values (Kd) of eIF4E binding affinity to the indicated RNA ligands. (E) The effect of eIF4E knockdown on GFP expression driven by mRNA with C or A as the initiating nucleotides. HEK293T cells were transfected with either eIF4E siRNA or a non-targeting siRNA (10 nM). 48 hr later cells were transfected again with GFP reporter genes driven either by RPL18 (C) or CMV (A) promoter. Cells were harvested 24 hr after the second transfection and analyzed by western blot with GFP, eIF4E and Tubulin antibodies as indicated. (F) HEK293T cells were transfected RPL18 and CMV driven GFP reporter gene. Six hours later, increasing amounts of 4EGI-1 were added to the media as indicated. Cells were harvested 24 hr after transfection and subjected to western blot using GFP and Tubulin antibodies.DOI: http://dx.doi.org/10.7554/eLife.21907.008

Fig 4: The inhibition of the mTOR/eIF4E pathway attenuate the generation of W>F substitutants in GC. A) A diagram illustrating the site of action of each inhibitor. B) HGC27 cells stably expressing tGFP‐F26W were culture in the normal, IFN‐γ or ‐W medium and treated with 4EGI‐1 (25 µM) for 24 h. Flow cytometry analysis for the tGFP signal intensity in each group. C) Western blot results showing the level of IDO, T‐mTOR, p‐mTOR, T‐eIF4E, and p‐eIF4E in HGC27‐tGFP‐F26W cells treated with IFN‐γ (or control) and various inhibitors as indicated. The eFT508 treatment was performed at two different concentrations (2.5 nM and 10 nM). D‒F) HGC27 and MKN45 cells with tGFP‐F26W stable expression were cultured in medium with Ctrl or IFN‐γ, and treated with Torin 1 or eFT508 (2.5 nM for low concentration and 5 nM for high concentration). Then, MG132 (10 µM) was added to inhibit proteasome and cells were sent for clow cytometry analysis. Schematic flow chart of the experimental design (D). Flow cytometry results for the signal intensity of tGFP in HGC27 (E) and MKN45 (F) cells. G, H) HGC27 (G) and MKN45 (H) cells stably expressing tGFP‐F26W were transfected with control siRNA or EIF4E siRNAs and cultured in control, IFN‐γ or tryptophan‐depletion (‐W) medium. Flow cytometry results showing the signal intensity of tGFP. I) A scheme of the influence of IFN‐γ‐mediated Trp consumption in the generation of W>F substitutants in EBV‐positive GC with mTOR/eIF4E pathway hyperactivation (left panel). Inhibiting mTOR/eIF4E pathway with inhibitors were demonstrated to alleviate the generation and presentation of W>F peptides. Data are presented as the mean ± SD. **P < 0.01, ns = not significant.

Fig 5: Effect of eIF4E overexpression on mouse microglia in immunofluorescence assay. A, Immunofluorescence staining of eIF4E and Iba-1 in hippocampal CA3 region of mice in each group; the arrow indicates the activation state of microglia. B, Comparison of the fluorescence intensity of eIF4E protein expression in the hippocampal CA3 region of mice in each group. C, Comparison of the fluorescence intensity of the expression of the microglial activation marker protein Iba-1 in the hippocampal CA3 region of mice in the two groups. D, Colocalization of eIF4E and microglia in the hippocampal CA3 region of the two groups of mice. E, Analysis of colocalization data of eIF4E and microglia in the hippocampal CA3 region of the two groups of mice. F, G, Western blot assay, expression of Iba-1 protein in the hippocampus of control and eIF4Eki/ki mice. H, qPCR assay. Oxytocin mRNA expression levels in the hippocampus of control and eIF4Eki/ki mice. I, eIF4E overexpression causes autism-like social cognitive impairment. Mann–Whitney U test was used for comparison between the two groups. The data are presented as mean ± standard deviation, and statistical significance is denoted as *p < 0.05 and **p < 0.01. In the study, n refers to the number of animals, with five acquisitions from each (hippocampus) slice, with a maximum of three slices obtained from each experimental animal used for each protocol (3 animals in each group).