Fig 2: Expression analysis of key downstream targets of mTOR complex 1 (mTORC1) signaling; 4E-BP1 and S6R in the placenta on 13 d.p.c. (A) Representative Western blot images of each target. (B) Comparison of relative density between Group C and Group L. Data represent the mean ± SE. Group C, control mice; Group L, L-NAME-induced FGR mice; 4E-BP1, eIF4E-binding protein 1; S6R, ribosomal S6 protein; MW, molecular weight. * p < 0.05.

Fig 3: Brain MRI, histopathology, and phospho-S6 immunostaining. Top row is patient III:6 and bottom row is patient III:7. (A and D) are axial T2-weighted MRIs at age 6 and 14 weeks, respectively, showing multiple areas of cortical thickening and blurring of the gray white junction often maximal at the bases of deep abnormal sulci throughout the right hemisphere in patient III:6 and restricted to the right posterior quadrant in patient III:7 (white arrows). (B and E) are low- and high-power hematoxylin- and eosin-stained images, respectively, of resected cortex showing cortical dyslamination with clusters of dysmorphic cytomegalic neurons (black arrow) consistent with FCDIIA. (C and F) are images showing positive phospho S6 immunostaining (Ser235/236, Cell Signaling #2211, rabbit polyclonal, 1:200) in dysmorphic cytomegalic neurons (black arrows), consistent with mTOR pathway activation. In comparison, (G) shows virtually absent phospho S6 immunostaining in control post mortem human cortex (scale bar B, 400 µm; F, 80 µm; C, E, and G, 200 µm).

Fig 4: DPS-2 acts as a dual inhibitor of MEK/ERK and PI3K/AKT kinase pathways.(A) Western blot analysis of protein levels of p-MEK and p-ERK kinases after treatment with 2 µ? and 5 µ? of DPS-2 for 1 hour and 24 hours in RKO, HT29, and Colo-205 cell lines. PLX4720 was used as a positive control for MEK/ERK pathway inhibition. Protein levels were normalized against tubulin. (B) Western blot analysis of protein levels of p-Akt 473, p-Akt 308, p-SAPK/JNK, p-p38, and p-S6R after treatment with 2 µ? and 5 µ? of DPS-2 for 1 hour and 24 hours in RKO, HT29, and Colo-205 cell lines. Protein levels were normalized against actin. (C) Effect of DPS-2on phosphorylation in melanoma cell lines. The inhibition of pMEK, pERK, and pAKT by DPS-2 was examined in cell lines with BRAF V600E and NRAS Q61K mutations. Cell lines were treated with increasing concentrations of DPS-2 (0-10 µM) for 24 hours. The results are shown as % of activity in comparison with untreated controls (DMSO).

Fig 5: Validation of analogue-specific S6K1 mutation AMyc-WT-S6K1 and myc-AS-S6K1 were transfected in HEK293 cells with HA-RpS6WT or HA-RpS65A. In vivo kinase assay was performed in the presence of 6-Bn-ATP-?-S. After immunoprecipitation using an anti-HA antibody, the thiophosphorylation of RpS6 was revealed by Western blot using an anti-thiophosphate ester antibody. Expression level of myc-WT-S6K1 and myc-AS-S6K1 or total S6K1 was revealed by Western blot on total extracts using the indicated antibody.B, CHEK293 or U2OS cells stably expressing myc-WT-S6K1 (WT) or myc-AS-S6K1 (AS) were transfected with tagged forms of S6K1 substrates (CAD, eIF4B, eEF2K, SKAR, IRS1, RpS6, PDCD4) or PRAS40. In vivo kinase assay was performed in the presence of 6-Bn-ATP-?-S. After immunoprecipitation, the thiophosphorylation was revealed by Western blot using an anti-thiophosphate ester antibody. * indicates unspecific band.