Fig 2: Knockdown of FUT1 is associated with decreased mTORC1 activity. (a) Representative immunoblots and quantitative analysis of mTOR activity in control and FUT1 knockdown cells. Lysates of MCF-7 and T47D cells transfected with control or FUT1 siRNAs were subjected to immunoblot analysis with antibodies against phospho-p70 S6K (Thr389) and total S6K. The resulting immunoblot bands were then quantified and computed as phospho-p70 S6K/total S6K ratio for mTOR activity. Values shown are mean fold change±S.E.M. of three independent experiments (*P<0.05). (b) Time course of mTORC1 activity in control and FUT1 knockdown MCF-7 cells. mTOR activity was determined by immunoblot analysis for the phosphorylation status of p70 S6K from 48 to 120 h after FUT1 knockdown. Levels of LAMP-1, LAMP-2 and LC3-II were analyzed in parallel over the indicated time points and actin was used as a loading control. Results are representative of two independent experiments

Fig 3: Role of constitutively active 5-HT6 receptors in increased mTOR activity in prefrontal cortex of Nf1+/- mice. (A). Representative Western blot assessing neurofibromin expression in PFC of adult WT and Nf1+/- mice. Data represent the ratios of immunoreactive signals of the anti-neurofibromin antibody to the immunoreactive signal of the anti-ß-actin antibody and are expressed in % of values in WT mice. (B). Representative Western blots assessing p70S6K phosphorylation at Thr421-Ser424 as an index of mTOR activity in PFC of adult WT and Nf1+/- mice. Data represent the ratios of immunoreactive signals of the anti-phospho-Thr421-Ser424-p70S6K antibody to the immunoreactive signal of the anti-p70S6K antibody and are expressed in % of values in WT mice. In (A,B), the data illustrated are the mean ± SEM of results obtained in three mice for the WT genotype and five mice for the Nf1+/- genotype. * p < 0.05, unpaired t-test. (C). Western blots assessing p70S6K phosphorylation at Thr421-Ser424 in PFC of Nf1+/- mice injected with either vehicle (n = 3) or Rapamycin (Rapa, 10 mg/kg, n = 3), or SB258585 (SB, 2.5 mg/kg, n = 3) or CPPQ (2.5 mg/kg, n = 3) 30 min before sacrifice. Data are expressed as in B. * p < 0.05, one-way ANOVA followed by Dunnett’s test. n.s.: not significant.

Fig 4: MDM4 expression affects mTOR activation.a Pathway analysis of mTOR signaling in SK-OV-3 cells. Red and green colors underlie proteins differently expressed in MDM4- compared with Empty vector-SK-OV-3 cells (red = upregulated, green = downregulated proteins). b WB of Empty Vector or MDM4-SK-OV-3 cells showing S6K1 and p70S6K1T389 expression (left panel) and quantification (right panel) (n = 3, one-sample t test p < 0.001). c WB of indicated proteins in tumor nodules (one per mouse) generated by IP injection of Empty Vector or MDM4-SK-OV-3 cells in mice; lower panels show quantification (n = 7 mice, two-sided Student’s t test, ***p < 0.001, *p = 0.048). d SunSet assay of Empty Vector- and MDM4-SK-OV-3 cells treated with rapamycin for 6 hours; quantification of two independent assays (right panel). e WB of indicated proteins in whole-cell extract (WCE) and co-immunocomplex from SK-OV-3 cells grown in EBSS for 1 hour and then in absence or presence of complete medium for additional 1 hour (CM). 1 mg of WCE was immunoprecipitated with anti-MDM4 antibody (IPaMDM4) or control Ig (IPCTL). Right panel shows the analysis of 1/100 of WCE.

Fig 5: The effect of proinflammatory cytokines and mTOR signaling 2 days following TAA induction. (A) Representative pictures of the immunohistochemical study in aortic segments 2 days post-TAA induction. The slides were stained with TNF-a, IL-6 and IL-1ß. The anti-rabbit horseradish peroxidase/diaminobenzidine detection system was used to visualize the expression (brown staining). Scale bar, 50 µm. Sham: NaCl-treated group (n=6). TAA: CaCl2-treated group (n=6). (B) Quantitation of the protein content of TNF-a, IL-6, and IL-1ß was performed by computerized planimetry in the aortic adventitia and aortic media in immunohistochemically stained slides. Data are presented as the mean ± standard error of mean, *P<0.05 vs. the Sham group. (C) Representative western blots demonstrated the upregulation of phosphorylation of proteins involved in the mTOR signaling pathway in the CaCl2-treated segment of aorta as early as 2 days, including phospho-mTOR, phospho-p70 S6K and phospho-S6. TNF-a, tumor necrosis factor-a; IL, interleukin; TAA, thoracic aortic aneurysm; IOD, integral optical density.