Journal: Theranostics

Article Title: TFE3, a potential therapeutic target for Spinal Cord Injury via augmenting autophagy flux and alleviating ER stress

doi: 10.7150/thno.46566

Figure Lengend Snippet: 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.

Article Snippet: Primary antibodies against Beclin1 (Cat. No. 3738), ATG5 (Cat. No. 12994), ATP6V1B2 (Cat. No. 14617), Ubiquitin (Cat. No. 3936), ATF4 (Cat. No. 11815), CHOP (Cat. No. 2895), AMPKα (Cat. No. 5832), p-AMPKα (Cat. No. 2535) p-FOXO3a (Cat. No. 9466), p-EIF2α (Cat. No. 3398), p-4EBP1 (Cat. No. 9456), mTOR (Cat. No.2983), p-mTOR (Cat. No.5536) and CARM1 (Cat. No. 4438) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activity Assay, Western Blot, Translocation Assay, Cell Culture

Journal: PLoS ONE

Article Title: Rapamycin Inhibits IGF-1-Mediated Up-Regulation of MDM2 and Sensitizes Cancer Cells to Chemotherapy

doi: 10.1371/journal.pone.0063179

Figure Lengend Snippet: (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.

Article Snippet: Antibodies specific for PARP (#9542), phospho-AKT (Ser473; #9271), total AKT (#9272), Phospho-S6 Ribosomal Protein (serine 235/236; #2211), S6 Ribosomal Protein (#2217), S6 Kinase (#9202), eIF4E, Phospho-4EBP1 (Ser65; #9456) or 4EBP1 (#9452) were purchased from Cell Signaling Technology and used at 1∶1000 dilutions.

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: The mTOR effectors 4EBP1 and S6K2 are frequently coexpressed, and associated with a poor prognosis and endocrine resistance in breast cancer: a retrospective study including patients from the randomised Stockholm tamoxifen trials

doi: 10.1186/bcr3557

Figure Lengend Snippet: Survival among systemically untreated patients in Stockholm 3 in relation to p4EBP1_S65 and 4EBP1 protein. Kaplan–Meier curves and multivariate Cox regression of distant recurrence-free survival (DRFS) and breast cancer survival (BCS) among systemically untreated patients in the Stockholm 3 cohort in relation to (a) p4EBP1_S65 cytoplasmic protein, (b) p4EBP1_S65 nuclear protein, (c) 4E-binding protein 1 (4EBP1) nuclear protein, (d) 4EBP1 nuclear protein in the progesterone receptor (PgR)-positive subgroup, (e) 4EBP1 protein cytoplasm ≥ nucleus, and (f) 4EBP1 protein cytoplasm ≥ nucleus in the PgR-positive subgroup. The Cox analysis included the following variables: adjuvant tamoxifen treatment, tumour size, Nottingham histological grade, oestrogen receptor alpha, and human epidermal growth factor receptor 2 (HER2) status. *For the PgR-positive/BCS multivariate analyses, HER2 was divided into four groups due to few events. CI, confidence interval; HR, hazard ratio.

Article Snippet: The slides were incubated with primary antibodies for 4EBP1 (53H11/#9644, diluted 1:100; Cell Signaling Danvers, MA, USA) or p4EBP1_S65 (174A9/#9456, diluted 1:100; Cell Signaling) overnight at 4°C.

Techniques: Binding Assay

Journal: Breast Cancer Research : BCR

Article Title: The mTOR effectors 4EBP1 and S6K2 are frequently coexpressed, and associated with a poor prognosis and endocrine resistance in breast cancer: a retrospective study including patients from the randomised Stockholm tamoxifen trials

doi: 10.1186/bcr3557

Figure Lengend Snippet: Distant recurrence-free survival among patients in Stockholm 3 with ER-positive/PgR-positive tumours treated with tamoxifen in relation to p4EBP1_S65 and 4EBP1 protein expression. Kaplan–Meier curves and multivariate Cox regression of distant recurrence-free survival (DRFS) among patients in the Stockholm 3 cohort with ER-positive/PgR-positive tumours, treated with tamoxifen, in relation to (a) p4EBP1_S65 cytoplasmic protein, (b) p4EBP1_S65 nuclear protein, (c) 4E-binding protein 1 (4EBP1) cytoplasmic protein, (d) 4EBP1 nuclear protein, and (e) 4EBP1protein cytoplasm ≥ nucleus. The Cox analysis included the following variables: tumour size, Nottingham histological grade, and human epidermal growth factor receptor 2 (HER2) status. *For the 4EBP1 analysis, HER2 was divided into four groups due to few events. BCS, breast cancer-specific survival; CI, confidence interval; ER, oestrogen receptor alpha; HR, hazard ratio; PgR, progesterone receptor.

Article Snippet: The slides were incubated with primary antibodies for 4EBP1 (53H11/#9644, diluted 1:100; Cell Signaling Danvers, MA, USA) or p4EBP1_S65 (174A9/#9456, diluted 1:100; Cell Signaling) overnight at 4°C.

Techniques: Expressing, Binding Assay

Journal: Journal of Hematology & Oncology

Article Title: Histone deacetylase inhibitor potentiated the ability of MTOR inhibitor to induce autophagic cell death in Burkitt leukemia/lymphoma

doi: 10.1186/1756-8722-6-53

Figure Lengend Snippet: Valproic acid combined with temsirolimus induced Burkitt leukemia/lymphoma (BL) cell autophagy. (A) As detected by flow cytometry, co-treatment of valproic acid (VPA, 0.5 mM) with temsirolimus (TEM, 1nM) increased LC3 intensity in BL cell lines Namalwa, Raji, Daudi and Ramos. (B) Western blot analysis revealed that combined treatment increased BECN1 expression, but decreased expression of P62, p-MTOR, p-4EBP1, p-P70S6K and MYC. (C) Ultrastructural study of Namalwa and Raji cells showed that combined treatment induces more frequently BL cell autophagy than each agent alone. (D-F) Growth inhibition of Namalwa cells was significantly reduced by autophagy inhibitor 3-Methyladenine (0.5 mM) and Bafilomycin A1 (10 nM) (D) , **P<0.01, *P<0.05 comparing with no inhibitor), by molecular silencing of the ATG5 siRNA ( E , *P<0.05 comparing with CON siRNA), but not by pan-caspase inhibitor ZVAD-FMK (0.3 mM) (F) .

Article Snippet: Antibodies against LC3-I/II (4108), phosphorylated MTOR (p-MTOR) (2971), MTOR (2972), phosphorylated 4E binding protein-1 (p-4EBP1) (9456), 4EBP1 (9644), phosphorylated P70 ribosomal S6 kinase (p-P70S6K) (9205), P70S6K (2708), HDAC3 (3949), HDAC4 (5392), phosphorylated AKT (p-AKT) (4060), AKT (9272), ACTB (4970), c-caspase-3 (9664), c-PARP (9541) and chemiluminescence phototope-horseradish peroxidase kit (7003) were obtained from Cell Signaling.

Techniques: Flow Cytometry, Western Blot, Expressing, Inhibition

Journal: Journal of Cancer

Article Title: Glycolysis is suppressed by DCZ0801-induced inactivation of the Akt/mTOR pathway in Multiple Myeloma

doi: 10.7150/jca.45146

Figure Lengend Snippet: DCZ0801 regulated mTOR kinase activity. OCI-MY5 and RPMI-8226 were cultured with 60 μM and 120 μM DCZ0801 for 48 h. Western Blot analysis was used to evaluate the protein expression level of Akt, p-Akt, mTOR, p-mTOR, p70s6k, p-p70s6k, 4E-BP1, p-4E-BP1 and Actin.

Article Snippet: Antibodies against cleaved caspase-8 (#9496), caspase-9 (#9508), caspase-3 (#9662), CDK2 (#2546), cdc25A (#3652), cyclinA2 (#1547), Akt (#9272), p-Akt (Ser473, #9271), p-4E-BP1 (Ser65, #9456), 4E-BP1 (#9644), p-mTOR (Ser2481, #2974), mTOR (#2983), p-p70S6K (Thr389, #9205), p70S6K (#2708), p-ERK1/2 (Ser383, #9181), STAT3 (#9139), PKM2 (#4053) and β-actin (#3700) were purchased from Cell Signaling Technology (Beverly, USA).

Techniques: Activity Assay, Cell Culture, Western Blot, Expressing