Fig 2: Detection of Singly and Doubly Phosphorylated 4EBP1. Control sample was prepared using C18 purified peptides from a tryptic digest (200 µg) of MKN45 cells (untreated). An Affi-BAMS assay for 4EBP1 (pT37 and pT46) was performed as described in the Methods section. An in silico tryptic digest of 4EBP1 produces the following peptides with either a single phosphorylation (at T37 or T46) or dually phosphorylated peptide (at both T37 and T46) with calculated masses (MH+, monoisotopic) for each of the peptides as 3127.498, 3207.465, 3283.599 and 3363.566 m/z (z = 1, MH+), respectively. The data collected from the MALDI MS spectrum shows the following measured peptide masses (interpolated monoisotopic) as 3126.78, 3206.63, 3283.08 and 3363.09 m/z (z = 1, MH+), respectively. These masses represent the 4EBP1 peptides corresponding to the zero and one missed cleavage product, with the one missed cleavage product corresponding to an additional arginine residue at the N-terminus of the resulting tryptic peptide (delta mass of 156.18 m/z). An 80 m/z mass shift is observed for both cleavage products, corresponding to the singly and doubly phosphorylated forms of each tryptic peptide since the antibody displays cross-reactivity with singly phosphorylated pT37 or pT46.

Fig 3: Elevated free amino acids during HF treatment is suggested to enhance mTORC1 signaling. (A) MEF cells were treated with 100 nM HF for 6 h in presence or absence of 25 nM Torin1 or left untreated (NT), followed by immunoblot analyses with the indicated antibodies. (B) The bar graph shows the changes in the levels of phosphorylated GCN2, eIF2a, S6K1, and 4EBP1. Results were derived from 3 independent experiments, with error bars represented as the means ± SD. Significant differences between treatment groups are indicated: *P < 0.05; **P < 0.005; ****P < 0.0001. (C) MEF cells were treated with 100 nM HF for 6 h or left untreated (NT), followed by lysis and free amino acids were measured and illustrated in the bar graph. The data was derived from 3 independent experiments, with error bars represented as the means ± SD. Significant differences between treatment groups are indicated: *P < 0.05; **P < 0.005; ***P < 0.0005. (D) MEF cells were treated with 2 mM histidinol for 6 h in presence or absence of 25 nM Torin1 or left untreated (NT), followed by immunoblot analyses with the indicated antibodies. (E) The bar graph shows the changes in the levels of phosphorylated GCN2, eIF2a, S6K1 and 4EBP1. The data was derived from three independent experiments, with error bars represented as the means ± SD. Significant differences between treatment groups are indicated: *P < 0.05; **P < 0.005; ****P < 0.0001. (F) WT and Gcn2–/– MEF cells were treated with either 50 or 100 nM HF for 6 h in presence or absence of 25 nM Torin1 or left untreated (NT), as indicated. Fresh media was then applied to cells for 18 h for a total of 24 h. Cell viability was monitored by the MTT assay. The data was derived from three independent experiments, with error bars represented as the means ± SD. Significant differences between treatment groups are indicated: *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

Fig 4: Isopsoralen (ISO) supresses the adipogenic and promoted osteogenic differentiation of bone-derived marrow mesenchymal stem cells (BMSCs) by inhibiting the activation of the mTORC1 pathway in vitro. (A) ISO altered the expression of osteocalcin (OCN) (G) and runt-related transcription factor 2 (RUNX2) (H) during the osteoblastic differentiation of cultured BMSCs for 7 days. Under adipogenic differentiation conditions for 1 week, ISO inhibited peroxisome proliferator-activated receptor ? (PPAR?) (C and K) and CCAAT/enhancer binding protein ß (C/EBPß) expression (C and L). PPAR? (E and O) and C/EBPß (E and P) expression was markedly decreased in the presence of ISO at day 14 in a dose-dependent manner (O and P). mTORC1 signaling was down-regulated in (B) the osteoblastic differentiation process and (D and F) under adipogenic conditions. (B and I) Eukaryotic translation initiation factor 4E-binding protein 1 (4E/BP1; Thr37/46) expression was upregulated and (B and J) phospho-S6 ribosomal protein (P-S6; S235/236) expression was decreased 2 weeks following osteogenic induction. The expression of (M and Q) 4E/BP1 (Thr37/46) and (N and R) P-S6 (S235/236) in adipogenic medium at (M and N) 7 days or (Q and R) 14 days was similar to that in BMSCs cultured under osteogenic conditions for 7 days. *P<0.05, **P<0.01 and ***P<0.001 compared to the group without ISO (Con, control). Columns represent the means ± SD from 9 wells/group (G–R).

Fig 5: Intracellular VH Domain expression disrupts eIF4F complex formation and cap-dependent translation in vitro.a Western blot analysis of 293FT cells transfected for 48 h with FLAG tagged empty vector (Mock), 4EBP1 and anti-eIF4e VH mutants, in whole cell lysate (left, WCL) and FLAG immunoprecipitation analysis (right). b The NanoBit assay consists of a split luciferase fused either to eIF4E or eIF4G606–646, whose interaction restores luciferase function41,74. Exogenous expression of antagonists of the eIF4E:4G interaction result in a decrease in total luminescence. SmBiT-eIF4E and eIF4G604–646-LgBiT constructs were co-transfected into HEK293 cells with either empty vector (Mock) or vectors containing 4EBP1 or VH mutants (as indicated) for 48 hrs. After 4 h of incubation, the luciferase activities of the reconstituted NanoBit protein were measured in living cells as described in Material and Methods. All values represent mean ± standard deviation (n = 3, independent experiments). c A bicistronic luciferase reporter (left panel), which measures the relative amount of cap-dependent translation (Renilla) to cap-independent translation (Firefly), was co-transfected with indicated plasmid. Renilla and Firefly luciferase activity was measured 48 h post transfection and plotted as a ratio-metric value. The Renilla luciferase is controlled by a GC rich 5’UTR whose activity correlates to cap dependent translation, whilst the Firefly luciferease is regulated by an internal ribosomal site (IRES). Lysates were prepared from the transfected cells and blotted for the presence of the respective constructs (below left-hand graph). In the right-hand graph, the lysates from the bicistronic assay were probed for eIF4E serine 209 phosphorylation eIF4E with the Alpha Surefire assay (Perkin-Elmer). This assay is a no wash sandwich bead-based immunoassay that enables quantification of eIF4E phosphorylation levels. All values represent mean ± standard deviation (n = 3, independent experiments). d Hela cells were transfected with indicated DNA using different amounts (µg) for 48 h and levels of indicated protein measured with western blot. Protein loading was assessed with ß-actin antibody. E) Lysates from the transfected HELA cells in d were also used to perform m7GTP pull down followed by western blot analysis. Levels of 4E-BP14ala and VH domain mutants were detected using anti-FLAG. All western blot analyses were repeated twice to verify their reproducibility. Representative blots are shown. Source data for a, b, c, d and e are provided in the Source Data file. Center line and limits denoted on dot blots represent the mean value and standard deviation of the replicates, respectively.