Fig 2: MNK kinase activity inhibition on eIF4E1 and eIF4E3.(a) Western blot showing total and phospho-eIF4E1(S209), MCL-1 and eIF4E3 in HLY-1 and Pfeiffer DLBCL cell lines after 48 h treatment with MNK inhibitors, cercosporamide and CGP57380. (b) Densitometry analysis showing band intensity relative to GAPDH of phospho-eIF4E1(S209), total eIF4E1, MCL-1 and eIF4E3 proteins in Fig. 4a. Mean±s.e.m., n=3, *P-value of Student’s t-test <0.05. (c) Trypan blue exclusion assay of HLY-1 (black bars) and Pfeiffer (white bars) cells after 48 h treatment with vehicle, 5 µM cercosporamide or 20 µM CGP57380, and values normalized to vehicle. Mean±s.d., n=3, *P-value of Student’s t-test <0.05. (d) Western blot analysis following cap-pull down of HLY-1 and Pfeiffer cells following 48 and 72 h treatment with MNK inhibitors, probed for total eIF4E1. (e) Densitometry analysis showing band intensity relative to GAPDH of total eIF4E1 after 48 h (left panel) and 72 h (right panel) treatment with 5 µM cercosporamide or 20 µM CGP57380, as in Fig. 4d. Mean±s.e.m., n=3, *P-value of Student’s t-test <0.05. (f) RT-qPCR analysis measuring total eIF4E3 mRNA level in HLY-1 cells, 24 or 48 h after treatment with CGP57380 (20 µM). Values are mean±s.e.m., n=3, *P-value of Student’s t-test <0.05). (g) Western blot showing eIF4E3 level in HLY-1 cells after 48 h treatment with 20 µM CGP57380 and/or 100 µg ml-1 cycloheximide. (h) Densitometry analysis showing band intensity relative to GAPDH of eIF4E3 level in HLY-1 cells after 48 h treatment with 20 µM CGP57380 and 100 µg ml-1 cycloheximide, alone or in combination, as in Fig. 4f. Mean±s.e.m., n=3, *P-value of Student’s t-test <0.05. Full immunoblots are shown in Supplementary Fig. 10.

Fig 3: 4E-BP2 and eIF4G association with eIF4E.(A) Samples as in Fig. 1A, were bound to m7GTP-Sepharose and analyzed by western blotting with anti-eIF4E (eIF4E), anti-4E-BP2 (4E-BP2), anti-phospho-4E-BP2 Thr37/Thr46 (p-Thr37/46) and anti-eIF4G (eIF4G) antibodies. Arrows show the relative position of eIF4E, the a and b forms of 4E-BP2, and eIF4G. The right numbers represent the apparent MW in kDa from protein markers. (B) Quantification of 4E-BP2 and eIF4G bound to eIF4E induced by ischemia and IR stress. No significant differences in the eIF4E levels were found (p = 0.360). Data are the quantification of bound 4E-BP2 (a + b forms; upper bar graph), or bound eIF4G (lower bar graph), with respect to eIF4E levels (ratios) detected with the corresponding antibody and represented in arbitrary units. Error bars indicate SEM. *p < 0.05, compared with the controls; + p < 0.05, +++p < 0.001, cerebral cortex, C, compared with the hippocampal CA1 region, CA1. Differences in eIF4G between R3d and SHC3d groups were done by two-tailed t test of data from six to eight different animals run in duplicate.

Fig 4: The phosphorylation of rpS6 is elevated in HL-60 macrophages and is sensitive to ERK1/2 signaling inhibition.(A and B) Western blots displaying the expression levels of rpS6 S235/236+phos, rpS6 S240/244+phos, total rpS6, 4E-BP1 T37/46+phos, total 4E-BP1, eIF4E S209+phos, total eIF4E and ß-Tubulin. The expression levels of these proteins were compared between HL-60 Mac, HL-60 PMN, A549 EC, KHYG-1 NK and H1299 EC cells, in panel A. Comparisons were also made between HL-60 cells treated with the solvent control, ERK1/2 signaling inhibitors (10 µM U0126 or 1 µM AZD6244), and mTOR inhibitor (100 nM Torin-1); in panel B. The band signal intensities were quantified and used to calculate the normalized band intensity ratios as described in the mathematical expressions. In panel B, “rpS6+phos fold change” was calculated based on the mathematical expression shown. [Control] and [Inhibitor] are defined as the rpS6+phos/rpS6 ratios of the control and inhibitor (Torin-1, U0126 and AZD6244) treatment cells, respectively, Experimental replicates were repeated over different days and the data from two repeats are shown.

Fig 5: Expression of promoters of cap-dependent translation initiation, and synaptic plasticity regulators in prefrontal cortex following 3 days active work (AW, collected at zeitgeber time, ZT0) and rest work (RW, collected at ZT12). (A,B) m7GTP pull-down analysis. (C) Representative immunoblots for (A,B). Blots normalized to total eIF4E in the corresponding immunoblot. Phospho-protein normalized to total protein in the corresponding immunoblot. (D,E) Western blot analysis. (F) Representative immunoblots for (D,E). Full-length blots/gels are presented in Supplementary Figure S2. Blots normalized to GAPDH in the corresponding immunoblots. Phospho-protein normalized to total protein in the corresponding immunoblot. Quantification of immunoblots are expressed as percentage change relative to time-matched undisturbed control (AWC collected at ZT0, RWC collected at ZT12; normalized to 100%). Plots show mean ± SEM, with scatter plot overlaid. N = 6–9/group. *p < 0.05, **p < 0.01, compared to time-matched control.