Fig 2: 4E1RCat or 4EGI-1 potentiated the effect of sorafenib by inhibiting eIF4E and eIF4G in HCC cell lines. (a) The western blot showed that eIF4E, eIF4G and phospho-4E-BP1 (Thr37/46) were downregulated after the combined administration for 48 h. (b) The transfection effectiveness of 4EBP1-Thr37/46 mutation was verified using western blot. (c) The MTT showed that the inhibition of phospho-4EBP1(Thr37/46) did not enhance the effect of sorafenib. (d) The PI3K-AKT-mTOR signaling pathway was inhibited after the coadministration of drugs for 48 h. The concentrations of sorafenib, 4E1RCat and 4EGI-1 were 8, 25 and 25 µM, respectively. All data were displayed as mean ± SD. 4E-BP1-over, 4E-BP1 overexpression; 4E-BP1-Thr37/46, 4E-BP1 Thr37/46 mutation; HCC, hepatocellular carcinoma; mTOR, mammalian target of rapamycin; NC, negative control.

Fig 3: Global changes of protein synthesis and eIF4F complex formation on cap after mechanical stimulation. (a) Representative images of SUnSET/Western blot analysis of new protein synthesis in tendon-derived stromal and C3H10T1/2 cells after mechanical stretching. (b) shows the densitometry analysis of anti-puromycin blots normalized to vinculin. (c) Lysates from non-stimulated and stimulated (mechanical stretching) human tendon cells were subjected to m7GTP pull-downs and analyzed for the indicated proteins by Western blot. Ratio paired t-test; mean ± SE; ns P > 0.05; * P ≤ 0.05; ***P ≤ 0.001; n ≥ 3 biological replicates.

Fig 4: Cardiac glycosides inhibit eukaryotic translation initiation factor 4A complex (eIF4A)-dependent translation in triple-negative breast cancer (TNBC) cells. (A). (CGG)4 Luc2-tdTomato readings in MDA-MD-231-S, MDA-MB-Bone-un, and SUM-159PT cells following treatment with 0-187.5 nM digoxin (12.5 nM increments). (B). Calculated (CGG)4 IC50 values for each cell line. (C). (CGG)4 Luc2-tdTomato/total protein readings (A595) in MDA-MD-231-S cells following treatment with either 0-150 nM digoxin or rocaglamide A. (D). CMV-Luciferase/RTK Renilla expression readings in 293 cells following treatment with 0–100 nM digoxin (* indicates p < 0.05 as analyzed by a one-way ANOVA with a Bonferroni post hoc test). (E). Western blots following treatment with 50 nM rocaglamide A for 48 h in SUM-159PT cells. Band intensities were calculated by densitometry based on the ratio of each target gene (BIRC5 or ROCK1) normalized to the β-actin protein signal with 0 nM set to 100. (F). Western blots following treatment with 0–50 nM digoxin for 48 h in SUM-159PT cells. Band intensities were calculated based on the ratio of each target gene (BIRC5 or ROCK1) normalized to the β-tubulin protein signal with 0 nM set to 100. (G). Western blots following treatment with 50 nM of each indicated drug for 72 h in SUM-159PT cells. Band intensities were calculated based on the ratio of each target gene (eIF4A, eIF4G, or eIF4E) normalized to the β-tubulin protein signal with 0 nM set to 100. Uncropped Blots are shown in Figure S2. (H). Quantitation of mRNA levels by qPCR from SUM-159PT cells following the treatment of 50 nM digoxin. Data were analyzed using the ΔΔCt method with β-tubulin as the control. Relative expression was calculated with DMSO-treated cells set to 100% (* indicates p < 0.05 as analyzed by a one-way ANOVA with a Bonferroni post hoc test).

Fig 5: Effects of MPA and NOMAc on protein levels of Akt/ mTOR/ 4EBP1/eIF4G in EC cells. (A-1,A-2,A-3,A-4) The expression levels of p-Akt/Akt, p-mTOR/mTOR, p-4EBP1/4EBP1 and p-eIF4G/eIF4G in wild-type RL95-2 cells. (B-1,B-2,B-3,B-4) The expression levels of p-Akt/Akt, p-mTOR/mTOR, p-4EBP1/4EBP1 and p-eIF4G/eIF4G in THRB-silenced RL95-2 cells. (C-1,C-2,C-3,C-4) The expression levels of p-Akt/Akt, p-mTOR/mTOR, p-4EBP1/4EBP1 and p-eIF4G/eIF4G in wild-type KLE cells. (D-1,D-2,D-3,D-4) The expression levels of p-Akt/Akt, p-mTOR/mTOR, p-4EBP1/4EBP1 and p-eIF4G/eIF4G in THRB-silenced KLE cells. The results are presented as mean ± SEM from three independent experiments. THRB, thyroid hormone receptor beta; si-Ctrl, negative control without THRB silencing; si-THRB, THRB silencing; T3, triiodothyronine; MPA, medroxyprogesterone acetate; NOMAc, nomegestrol acetate; Akt, phosphorylated protein kinase B; mTOR, mammalian target of rapamycin; 4EBP1, 4e-binding protein 1; eIF4G, eukaryotic translation initiation factor 4G.