Fig 2: The effects of coagulation factors on the expression and phosphorylation of mTOR and its downstream substrates in Hep3B cell line. (a) Hep3B cells were treated with TF (200 ng/ml), FVII (200 ng/ml), PAR2 agonist (200 ng/ml) for 24 h, and total amount of cells was harvested and detected for mTOR, p-mTOR, p70S6K, p-p70S6K, 4EBP1, p-4EBP1 and β-actin by western blot assay. (b) Hep3B cells were pre-transfected with mTOR siRNA (30 nM) for 24 h, respectively; subsequent treatment of each coagulation factor (TF, 200 ng/ml; FVII, 200 ng/ml; PAR2 agonist, 200 ng/ml) for 24 h and the western blot assay was performed for LC3A/B and β-actin. All results are expressed as the mean±S.D. from three independent experiments

Fig 3: The activity of TFE3 after SCI is regulated by AMPK-mTOR and AMPK-SKP2-CARM1 signaling pathways. (A) Western blot analysis of AMPK-mTOR signal pathway in the cytoplasm in spinal cords from the control mice, SCI mice, and SCI mice treated with DMSO or Compound C, at Day3. (B) Densitometric analysis of the AMPK, p-AMPK, p-mTOR and p-4EBP1 data from (A), normalized to the loading control GAPDH. (C) Western blotting of TFE3 nuclear translocation at the lesion for each group. (D) Corresponding densitometric analysis of the TFE3 bands in (C) normalized to the loading control H3. (E) Western blots of the AMPK-SKP2-CARM1 signaling pathway in the nucleus of the indicated groups at Day3 after SCI. (F) Densitometric analysis of AMPK, p-AMPK, p-FOXO3a, SKP2 and CARM1 bands from (E) normalized to control H3. (G) Nuclear CARM1-TFE3 complex was detected by IP in indicated groups at Day3 after SCI. (H) Densitometric analysis of TFE3 and CARM1 data from (G) normalized to loading control H3. (I) Western blot analysis of LC3, SQSTM1/p62 and UB in the spinal cord lesion of each group at Day3 after SCI. (J) Densitometric analysis of band data from (I) normalized to the loading control GAPDH. (K) Western blotting of LC3II in the indicated mice spinal cord slides cultured in the presence or absence of CQ at Day3. (L) Densitometric analysis of LC3II from (K) normalized to the loading control GAPDH. n=6, ns stands for not significant, *P<0.05, **P<0.01.

Fig 4: Effects of SLC6A14 on CRC via the activation of mTOR signaling. A The protein expression of mTOR signaling and MMPs in the HCT116 or Caco2 cells transfected with SLC6A14-siRNA or blockade of SLC6A14 with α-MT was analyzed by Western blot. B The relative protein levels of SLC6A14 and mTOR signaling molecules, and β-actin was used as the internal control. C The relative protein levels of MMP2, MMP9, and MT1-MMP, and β-actin was used as the internal control. D Western blot analysis was used to determine the downstream effectors of mTOR in HCT116 or Caco2 cells transfected with SLC6A14-siRNA or blockade of SLC6A14 with α-MT. E The relative protein levels of P-p70S6, P-S6 and P-4EBP1, and β-actin was used as the internal control. F Western blot analysis was used to determine the total-Akt and phosphorylated-Akt (Ser473) in HCT116 or Caco2 cells transfected with SLC6A14-siRNA or blockade of SLC6A14 with α-MT. G The relative protein levels of Akt and P-Akt, and β-actin was used as the internal control. The data are expressed as the mean ± SD of three independent experiments. *P < 0.05

Fig 5: Signaling downstream of mTOR is required for IGF-1-induced MDM2 up-regulation.(A) H1299 cells were transiently transfected with wild-type 4EBP1, 4EBP1-5A, or vector plasmid. Twenty-four hours after transfection, cells were split and maintained for an additional 24 hours prior to serum starvation for 24 hours. Cells were then treated with or without IGF-1 (50 ng/mL) for six hours. Cells were lysed and subjected to western blotting. Quantitative analyses of MDM2 protein levels were performed by densitometry scanning for each individual MDM2 protein band and the corresponding actin protein band. The ratio of MDM2 over actin in the control sample (lane 1) was arbitrarily set as 1.0. (B) U2-OS cells were transiently transfected with siRNA specific against eIF4E (si4E) or a scrambled control (siScr). Cell lysates were subjected to western blotting, as shown. (C) U2-OS cells transfected with si4E or siScr were serum-starved for 24 hours prior to treatment with or without 5 ng/mL IGF-1 for six hours. Cell lysates were subjected to western blotting, as shown. (D) U2-OS cells were transiently transfected with siRNA specific against S6K (siS6K) or a scrambled siRNA (siScr). Cell lysates were subjected to western blotting, as shown. (E) U2-OS cells transfected with siS6K or siScr were serum-starved for 24 hours prior to treatment with or without 5 ng/mL IGF-1 for six hours. Cell lysates were subjected to western blotting, as shown.