Fig 2: mTORC1 controls CTPS cytoophidium formation through S6K1. A: SW480 cells were transfected with siRNA targeting S6K1, ATF4 or MTHFD2, or scrambled control siRNA for 48 h, and cells were subjected to immunofluorescence staining with anti-CTPS antibody. B: Percentages of SW480 cells with CTPS cytoophidia shown in (A). C: Western blotting analysis of S6K1, ATF4 and MTHFD2 protein expression in cells transfected with siRNA targeting the indicated genes. D: SW480 cells infected with two distinct shRNA targeting S6K1 or RFP (as control) were analyzed by immunofluorescence staining for the presence of CTPS cytoophidium. E: Quantitative data of percentage of cells with CTPS cytoophidia shown in (D). F: Immunoblotting analysis of S6K1 expression in cells stably expressing shRNA targeting S6K1 or RFP.G?I: Overexpression of CA-S6K1 reverses mTOR knockdown-induced reduction of cytoophidium formation. SW480 cells expressing control or mTOR shRNA were infected with lentiviruses expressing HA-CA-S6K1. The resulted cells were subjected to immunofluorescence (G and H) and Western blotting (I) analysis with appropriate antibodies and the percentages of cells bearing cytoophidium were quantified (H). J: SW480 cells stably expressing HA-CA-S6K1 were cultured for 48 h and the lysates prepared were subjected to immunoprecipitation by anti-HA antibody or normal mouse IgG and the presence of CTPS was examined by immunoblotting using anti-CTPS antibody. ß-Actin and a-Tubulin were used as a loading control in (C and I) and (F), respectively. Mean ± S.E.M., n.s., not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001 versus control. Scale bars = 20 µm. One of four to six similar experiments is shown.

Fig 3: a-KG could reduce neuronal apoptosis by inhibiting IR. (a) Level of p-IRS1 (Ser636/639), IRS1, p-IRS1 (Ser636/639)/IRS1 ratio, and p-S6K1 (Thr389) in control, Con+PA, SAH, SAH+DMKG, and SAH+DMKG+PA groups (one-way ANOVA, F = 10.82; F = 10.76; F = 17.02; F = 15.73). (b) The ratio of GLUT4 expression in plasma membrane to total levels of GLUT4 expression in control, Con+PA, SAH, SAH+DMKG, and SAH+DMKG+PA groups (one-way ANOVA, F = 72.93). (c) Glucose uptake ability was calculated by the average fluorescent intensity of 2-NBDG in the control, Con+PA, SAH, SAH+DMKG, and SAH+DMKG+PA groups, bar = 200 µm (Kruskal-Wallis). (d) Expression of cleaved caspase-3 in the control, Con+PA, SAH, SAH+DMKG, and SAH+DMKG+PA groups (one-way ANOVA, F = 9.52). (e) Proportion of early apoptotic detected by Annexin V-FITC/PI staining (one-way ANOVA, F = 39.1) (*P < 0.05, **P < 0.01).

Fig 4: (a) The binding mode of alpha-ketoglutarate to S6K1 protein was obtained based on docking. The left picture is the overall view, and the right picture is the local view. Blue stick is the small ligand molecule, light blue cartoon is the protein, gray dotted line represents hydrophobicity, and yellow dotted line represents hydrogen bonding. (b) The ability of a-KG to inhibit S6K1 activity was assayed by ADP-Glo™ kinase kit (one-way ANOVA, F = 553.81) (**P < 0.01).

Fig 5: IPO7 regulates the activation of PI3K/AKT-mTOR pathway. (A,B) Gene set enrichment analysis (GSEA) shows the enriched gene expression signature of IPO7 and GTF2I in the TCGA datasets. (C,D) Immunoblots analysis shows the GTF2I levels in the nucleoplasm and cytoplasm after transfecting with shIPO7 or Importazole. (E) The correlation of GTF2I with PIK3CA and PIK3C2A. (F) The correlation of IPO7 with PIK3CA and PIK3C2A. (G) Immunoblot analysis of phosphor-mTOR (p-mTOR), mTOR, phosphor-AKT(p-AKT), AKT, phosphor-P70S6K (p-P70S6K), P70S6K expression in shCtrl, shIPO7, and Importazole groups. The relative expressions were quantified by normalizing to GAPDH. (H) Immunoblot analysis of phosphor-mTOR (p-mTOR), mTOR, phosphor-AKT(p-AKT), AKT, phosphor-P70S6K (p-P70S6K), P70S6K expression in siNC and siGTF2I groups. The relative expressions were quantified by normalizing to GAPDH. Error bars represent mean ± standard deviation (SD). *p < 0.05.