Fig 2: SGK3 expression in relation to (activated) signalling components of the PI3K/Akt system in primary MM cells.Shown are Western blots prepared from frozen pellets of primary MM cells purified by CD138 microbead selection. The material was used to load two gels, with the representative ß-actin control belonging to the same blot on which PI3K-p110a, P-FOXO1/3A, P-Akt (Thr308) and pan-Akt were also stained. Of note, the phospho-Akt (Thr308) antibody also stained a slightly larger band in most primary MM samples (marked by "?") that was not visible in any MM cell line. This band runs slightly higher, though, than the SGK3 band as stained on the parallel blot. Staining for P-PRAS40 (CST; no. 2997) was performed after staining for P-GSK-3ß. Since both antibodies were raised in rabbit the latter signal reappeared in the P-PRAS40 blot.

Fig 3: IR trafficking is impaired on class III PI3K inactivation.(a) Hepa1.6 cells were transduced with shRNA Vps15- or shRNA SCR-expressing adenoviruses. IRß-RFP was overexpressed 24 h postinfection and cells were imaged 24 h post-transfection. Representative spinning disc confocal snap shot image of the live cell used for endosome tracking is presented (the corresponding movie is available as a Supplementary Information File). Endosomes were manually tracked using MetaMorph software. Cumulative endosome velocity (b) and average endosome velocity (c) were calculated for each endosome. Data are means±s.d. (n=95–125 endosomes, *P<0.001 versus Adeno-shRNA SCR, Mann–Whitney Rank-sum test). (d) Hepa1.6 cells were transduced with shRNA Vps15- or GFP-expressing adenoviruses. The 24-h postinfection cells were serum starved for 24 h followed by stimulation with 1 µM insulin for 2 h. Before stimulation with insulin, cells were pretreated with 3 µM of PitStop2 inhibitor for 15 min. IRß levels and status of the pathway activation was determined by immunoblot analysis. Densitometric analyses of phosphorylated Akt and Pras40 normalized to levels of total proteins are presented on Supplementary Fig. 1e.

Fig 4: SLC6A14 depletion mediates amino acid starvation to alleviate the activation of the PI3K/AKT/mTORC1 pathway. (a) MKN28-M cells were transfected withLV-pTZU-SLC6A14 plasmid (MOI = 60); 48 h later, differentially expressed genes (DEGs) in SLC6A14-silenced-MKN28-M, compared to MKN28-M cells, were identified using mRNA microarray analysis and were shown in heat map. (b) KEGG pathway analysis was carried out to clarify the top 10 overrepresented canonical pathways that were enriched by these DEGs in MKN28-M cells following LV-pTZU-SLC6A14 transfection. (c–f) Indicated cells were treated with a-MT (5 mM), LV-pTZU-SLC6A14 (MOI = 60), or LV-pcDNA3.1-SLC6A14 (MOI = 60) plasmid for 48 h. Western blot analysis was conducted to detect the expression of signaling proteins in the PI3K/AKT pathway in (c) MGC-803, (d) BGC-823, (e) SGC7901-M, and (f) MKN28-M cells, respectively. (g–j) Indicated cells were treated in accordance with the methods as described in (c–f); the expression of mLST8, total, and phospho-Raptor, phospho-PRAS40, phospho-p70S6K, and phospho-4EBP1 was determined by Western blot analysis. For (c–j), the experiments were repeated for three times, and representative results were described as the representatives (upper panels) and the means ± SD of relative expression (bottom panels). *P < 0.05, **P < 0.01, and#P > 0.05. (k, l) GC cells were stimulated with a-MT (5 mM), LV-pTZU-SLC6A14 (MOI = 60), or LV-pcDNA3.1-SLC6A14 (MOI = 60) plasmid for 48 h. (k) ASNS and (l) CHOP mRNA were examined by Quantitative Real-time PCR assay. Data were displayed as the relative expression that were normalized to HPRT1 with the means ± SD from three irrelevant experiments. **P < 0.01.

Fig 5: Dynamic mTOR model.(A) Scheme summarizing the mTOR pathway. Amino acids directly activate mTORC1, which targets PRAS40-pS183, p70-S6K-pT389, and 4E-BP1-pT37/46. The insulin signal is transduced via the IR. The IR phosphorylates and recruits IRS1 which in turn recruits and activates PI3K. PI3K promotes the translocation of PDK1 to the membrane, where PDK1 phosphorylates Akt-pT308. Akt phosphorylates and inhibits the endogenous mTORC1 inhibitors PRAS40 at T246 and TSC2 at T1462. Thus, Akt activates mTORC1. p70-S6K inhibits IRS1 by direct phosphorylation at S636/639. PI3K also activates mTORC2, which targets Akt-pS473. In this study, we show that upon stress, mTORC1 is activated via the PI3K axis to promote stress granule assembly. (B) Topology of model I without stress input (Supplemental Data 1). Brown squares = species included in the model, circles = species variants (P, phosphorylation at the indicated site; cyt, cytosolic localization; mem, cell membrane localization; and *, active state), dark brown = observable species, species in ellipses = possible inputs to the model (insulin and amino acids) and inhibitory agents (MK-2206 and wortmannin), dark blue lines = mTORC2 activity, and light blue lines = mTORC1 activity. (C) Wortmannin fully inhibits PI3K (100%) in MCF-7 cells upon arsenite treatment. Akt-T308 phosphorylation (data shown in Fig S3A) was used to calculate the extent of PI3K inhibition upon wortmannin treatment. A one-way ANOVA was used to calculate P-values of Akt-T308 phosphorylation upon arsenite and arsenite + wortmannin treatment. As under wortmannin treatment, there was no significant induction of Akt-pT308 (P = 0.3226); the extent of inhibition for wortmannin was set to 100%. (D) MK-2206 partially inhibits Akt (83%) in MCF-7 cells upon arsenite treatment. TSC2-T1462 phosphorylation (data shown in Fig S3C) was used as to calculate the extent of Akt inhibition upon MK-2206 treatment. A one-way ANOVA was used to calculate P-values of TSC2-1462 phosphorylation upon arsenite treatment and arsenite + MK-2206 treatment. As under MK-2206 treatment, there was still a significant induction of TSC2-pT1462 (P = 0.0239), the extent of inhibition for MK-2206 was calculated by comparing the slopes of induction curves (83%) between carrier and MK-2206 treatment (indicated as red lines). (E) Summary of the extent of inhibition for wortmannin and MK-2206 in MCF-7 cells under arsenite treatment.