Fig 2: Multiple distinct mechanisms for downregulation of AKT activity in TSC1/2-deficient SCs. A-F: Western blots of sciatic nerve lysates from control and TSC2-SCKO/TSC1-SCKO mice at age P28 (A-E) or P14 (F) (3 mice per group), probed with the indicated antibodies. G: Immunofluorescence of longitudinal frozen sciatic nerve sections from control and TSC2-SCKO mice (age P28) showing greatly increased immunoreactivity of PTEN in the mutant (cytoplasmic signals surrounding DAPI+ nuclei). Scale bars: 50 µm H: Schematic summarizing distinct mechanisms underlying the downregulation of AKT activity in SCs from TSC1/2-SCKO nerves. Components highlighted in red are upregulated while blue highlighting depicts downregulation in mutant nerves. Note downregulation of insulin receptor substrate 1 (IRS-1) by constitutively active S6 kinases downstream of mTORC1. This leads to attenuated activation of PI3K/AKT. Lack of mTORC2 stimulation occurs through abolished TSC1/2 which results in reduced AKT phosphorylation in position Ser473 as well as through feedback inhibition by S6 kinases. Lastly, hyperactive mTORC1 promotes Hif1α transcription factor expression which results in increased PTEN transcription, and thus attenuated PI3K/AKT signaling.

Fig 3: Effects of Iturin A on STb-R-restricted mTORC1 pathway in semimembranosus muscle. (A) Western blotting analysis of IGF-1R, IRS-1, p-PI3K, p-Akt, p-mTOR, p-S6K1, p-S6, p-4EBP1, and eIF4E in semimembranosus muscle shows the change of mTORC1 signaling pathway after being fed with Iturin A and STb-R. (B) Quantification of the western blotting results and the values of the relative protein level are represented as the ratio of IGF-1R/β-actin, IRS-1/β-actin, p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR, p-S6K1/S6K1, p-S6/S6, p-4EBP1/4EBP1, and eIF4E/β-actin. Data are presented as mean ± SEM, and the different lowercase letters above standard error bars indicate significant differences, n = 3.

Fig 4: SNHG16 promotes hRMEC dysfunction via the PI3K/AKT pathway activation through IRS1Experiments were conducted in cells treated with LG (5 mM) for 48 h. (A) Quantitative real-time PCR showed the change of IRS1 level in SNHG16-overexpressed hRMECs by transfecting shRNAs for IRS1. Two shRNAs with relatively higher knockdown capacity were selected. n = 3 in each group. (B and C) CCK-8 assay and EdU assay showed the effects of IRS1 shRNAs and LY294002 on cell proliferation in SNHG16-overexpressed hRMECs. The shRNA with the highest knockdown capacity was adopted. n = 3 in each group. (D and E) Wound-healing assay and Transwell assay showed the effects of sh-IRS1#1 transfection and LY294002 addition on cell migration in hRMECs after SNHG16 overexpression. n = 3 in each group. (F and G) Quantitative real-time PCR and ELISA showed the effects of sh-IRS1#1 transfection and LY294002 addition on VEGF level in hRMECs after SNHG16 overexpression. n = 3 in each group. (H) Tube formation assay showed the effects of sh-IRS1#1 transfection and LY294002 addition on angiogenesis in SNHG16-overexpressed hRMECs. n = 3 in each group. All data were acquired from three independent experiments and presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01.

Fig 5: Compound 1 enhanced insulin-stimulated glucose uptake on C2C12 myotubes through activating insulin signaling pathway. (A) Cytotoxicity of compound 1 on C2C12 myotubes when treated for 24 h by MTT assay. (B) Compound 1 increased insulin-stimulated glucose uptake on C2C12 myotubes. AICAR was used as a positive control. (C) Compound 1 activated the insulin signaling pathway in C2C12 myotubes. The expressions of p-IRS-1, IRS-1, p-Akt, Akt, and GAPDH were analyzed by Western blots. “+” means presence, and “−” means absence. Data are expressed as mean ± SD, n = 6. # p < 0.0001, control vs. insulin; * p < 0.001, compound 1 or AICAR vs. insulin.