Fig 2: Construction and calibration of new mathematical models of the PI3K/MTOR signalling network incorporating the MLST8 (de)ubiquitination switch.(A) Schematic displaying a molecular switch mediated by MLST8 ubiquitination recently reported in [7], which dictates how MLST8 associates with mTORC1 or 2 through raptor and SIN1, respectively (ubi: ubiquitination). (B) Detailed reaction diagrams of the new PI3K/MTOR network models incorporating the MLST8 switch. Four model variants (models 1–4) with distinguishing interactions as described in the text are highlighted (ubi: ubiquitination, act: activated, Tot: total). (C) Simplified schematics of the different network structures captured by the four model variants. (D) Time-course model simulations using best-fitted parameter sets as compared to the quantified experimental data (red curves), presented for each of the four models. Experimental data represent the dynamic response of various network proteins following insulin stimulation (100nM) in MEF cells, reproduced from Western blot data representative of at least three biological replicates obtained from [7]. Per model, multiple best-fitted sets obtained from model calibration (n = 189, 198, 12, 13 for model 1, 2, 3, 4, respectively) were used for simulations: the lines represent the mean simulated curve and the shaded areas indicate standard deviation of all curves.

Fig 3: Experimental validation of IRS1/2-mediated regulation of MLST8 ubiquitination.(A) Flag-MLST8 immunoprecipitates from IRS1/2 knockdown C2C12 cells after insulin stimulation were analyzed by Western blot. (B) Graph shows mean ± S.E.M. of quantitative analyses of the Western blots in Fig 3A normalized for the total proteins (A.U., arbitrary units. * P<0.05, **P<0.01, two-tailed student’s t test, n = 3 biological replicates).

Fig 4: Interrogation of MLST8 alteration and prognostic value in cancer patients.(A) Alteration frequency of the MLST8 gene in cancer patients analyzed from TCGA database using cBioPortal (www.cbioportal.org), shown for the most frequently altered tumour types (cut-off > 1%). (B) Frequency of MLST8 alteration in breast cancer patients analyzed using two breast cancer cohorts TCGA and METABRIC. Only patients having MLST8 alterations are displayed for brevity. (C) Breast cancer patients with high MLST8 expression are significantly associated with poorer overall survival outcome compared to those with low MLST8 expression. (D) No significant differences in overall survival between patients having ‘low’ and ‘high’ expression of MAPKAP1, the gene encoding SIN1.

Fig 5: The regulation of ANT-AMPK-mTORC1 signaling pathway by ATR. (A) Immunoblot analysis results of ANT2, AMPK, phosphorylated AMPK (p-AMPK), Raptor, mLST8, mTOR, and phosphorylated mTOR (p-mTOR). (B) The relative protein expression in reference to GAPDH. Data are expressed as mean ± SD (n = 8). *p < 0.05, **p < 0.01, ***p < 0.001 (* represents HFD groups compared with the WT group). # p < 0.05, ## p < 0.01, ### p < 0.001 (# represents ATR treatments compared with the HFD group). WT = control group and HFD = high-fat diet group.