p 4ebp1 thr 37 46  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: Glycerol-3-phosphate acyltransferase-1 upregulation by O-GlcNAcylation of Sp1 protects against hypoxia-induced mouse embryonic stem cell apoptosis via mTOR activation

doi: 10.1038/cddis.2015.410

Figure Lengend Snippet: Involvement of mTOR in GPAT1-induced mESCs anti-apoptosis under hypoxia. ( a ) Cells were transfected with gpat1 and NT siRNA for 24 h before glucosamine (10 μ M) for 30 min. Subsequently, cells were exposed to hypoxia treatment for 24 h. Collected samples are lysed, and p-mTOR, GPAT1, and β -actin protein expressions were measured by using western blotting. Each result shown is representative of three independent experiments. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with glucosamine. ( b ) Cells were pretreated with rapamycin (10 nM) before glucosamine (10 μ M) treatment; and then, cells were exposed to hypoxia for 24 h. Total proteins were extracted, and blotted with Bcl-2, Bax, cleaved caspase-9, and β -actin. Each result shown is representative of three independent experiments. ( c ) Cells were pretreated with glucosamine and/or rapamycin (10 nM) for 30 min before hypoxia treatment, and cytochrome c, COX IV, and β -tubulin in the cytosolic and mitochondrial fraction were detected by western blot. ( d ) Cells were pretreated with various concentrations of LPA (10 − 6 M–10 − 9 M) before hypoxia treatment for 24 h. Cell viability was measured by trypan blue exclusion assay. Error bars are presented as a mean±S.E.M. of three independent duplex dishes. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone. ( e ) Cells were pretreated with pertussis toxin (100 ng/ml) for 30 min before LPA treatment (0.1 μ M) for 30 min. Subsequently, cells were exposed to hypoxia treatment for 24 h. Total proteins were extracted and blotted with p-mTOR, mTOR, p-S6K1, S6K1, p-4EBP1, 4EBP1, and β -actin. Each result shown is representative of three independent experiments. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. ( f ) p-mTOR was immunostained with p-mTOR antibody, and counter-stained with PI. Fluorescence images were acquired by using confocal microscopy. Fluorescence intensity of p-mTOR was quantified by using ImageJ software. Data are presented as a mean±S.E.M. of three independent experiments. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone. ( g ) Cells were pretreated with rapamycin (10 nM) before LPA (0.1 μ M) treatment; and then, cells were exposed to hypoxia for 24 h. Total proteins were extracted, and blotted with Bcl-2, Bax, cleaved caspase-9, cleaved caspase-3, and β -actin. Each result shown is representative of three independent experiments. ( h ) Cell viability was measured by using cell counter. Data are presented as a mean±S.E.M. of three independent duplex dishes. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. ( i ) Viable cells were measured by using annexin V/PI flow cytometry analysis. Annexin V-negative-PI-negative cells (Q3) were considered viable, annexin V-negative-PI-positive cells (Q1) were considered necrotic, annexin V-positive-PI-positive cells (Q2) were considered late apoptotic, and annexin V-positive-PI-negative cells (Q4) were considered early apoptotic. Data are presented as a mean±S.E.M. of two independent duplex dishes. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. * P <0.05 versus control, # indicates P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. The proposed model for signaling pathways involved in glucosamine-induced mESCs survival under hypoxia ( j )

Article Snippet: Mammalian target of rapamycin (mTOR), p-mTOR (Ser 2448), S6K1, p-S6K1 (Thr 389), 4EBP1, and p-4EBP1 (Thr 37/46) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Western Blot, Control, Trypan Blue Exclusion Assay, Staining, Fluorescence, Confocal Microscopy, Software, Flow Cytometry, Protein-Protein interactions

Journal: Biochemical pharmacology

Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

doi: 10.1016/j.bcp.2012.10.007

Figure Lengend Snippet: PI3K/mTORc1 and MAPK signaling are activated by GDF15 in ovarian cancer cells. (A) SKOv3 cells were serum starved overnight, and then stimulated with 20 ng/mL rhGDF15 for 2 or 5 min, or with the corresponding volume of vehicle control for 5 min. Western blots of total protein lysates were performed at least 3 times for p-Thr180/ Tyr182 p38MAPK, total p38, p-Thr202/Tyr204 p42/p44 Erk1/2, total Erk1/2, p-S473 Akt, and total Akt; representative blots are shown. Quantification is shown as a ratio of phospho-protein to total protein, and is shown relative to vehicle control. (B) SKOv3 cells were plated in matrigel. Cells were maintained in media containing vehicle control, 20 ng/mL rhGDF15, or GDF15 plus 1 µM PI3K inhibitor LY294002, 100 nM MEK inhibitor PD0325901, or 10 µM p38MAPK inhibitor SB203580. Media and drugs were changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. Fold change in anchorage-independent (AI) growth is shown relative to the vehicle control group; ** p < 0.005 for GDF15-stimulated versus control vehicle, and for inhibitor + GDF15 groups versus GDF15 alone. (C) Total protein lysates from SKOv3 stable control and GDF15 clones 1, 3, and 5 were Western blotted at least twice for phosphorylated and total p38MAPK, Akt, and 4EBP1; representative blots are shown. Quantification is shown as a ratio of phosphorylated to total protein, and is shown relative to control clone. (D) (Left) GDF15 stable clone 1 (G1) and clone 3 (G3) and control empty vector clone cells were treated with 10 nM or 100 nM of rapamycin, or with (right) 1, 5, or 10 µM TGF beta receptor type II inhibitor SB431542. Control groups (C) were treated with DMSO alone. After 72 h, MTS proliferation assays were performed. Proliferation is shown as a percentage of the control vehicle group per cell line, and reflects the average of six replicates. (E) SKOv3 cells were plated in matrigel. Cells were maintained in media containing vehicle control, 20 ng/mL GDF15, or GDF15 plus 100 nM rapamycin or 5 µM TGF beta receptor type II inhibitor SB431542. Media and drugs were changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. Fold change in anchorage-independent (AI) growth is shown relative to the control group. (F) GDF15 stable clone 1 (G1) and clone 3 (G3) and control empty vector clone cells were treated with DMSO control (C) and the concentrations shown for LY294002, PD0325901, or SB203580. After 72 h, MTS proliferation assays were performed. Proliferation is shown as a percentage of the control vehicle group per cell line, and reflects the average of six replicates.

Article Snippet: Blots were probed overnight using the following antibodies: from Cell Signaling, monoclonal p-S473 Akt-XP (1:1000), and polyclonal antibodies against: total Akt (1:1000), p-Thr202/Tyr204 p42/p44 Erk1/2 (1:1000), total p42/p44 Erk1/2 (1:1000), p-Thr180/Tyr182 p38 XP (1:1000), p-Thr 37/46 4EBP1 (1:1000), total 4EBP1 (1:1000), N-Cadherin (1:1000).

Techniques: Control, Western Blot, Clone Assay, Stable Transfection, Plasmid Preparation

Journal: Biochemical pharmacology

Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

doi: 10.1016/j.bcp.2012.10.007

Figure Lengend Snippet: GDF15 knockdown reduces invasion and growth in association with reduced p-4EBP1 in GDF15-overexpressing ovarian cancer cells. (A) Real-time PCR was performed for GDF15 in SKOv3 and Tov21 cells, and normalized to the level of internal control transcript RPLPO. Values reflect the average fold change in normalized GDF15 transcript. (B) Tov21 cells were infected with lentiviral GDF15 shRNA or control shRNA. Real-time PCR was then performed for GDF15 and RPLPO. Values reflect the average fold change in normalized GDF15 transcript. (C) Tov21 cells were infected with lentiviral GDF15 shRNA or control shRNA. Real-time PCR was performed for MMP-2, MMP-9, and VEGF, and normalized to RPLPO. Values reflect the average fold change in normalized transcript. (D) Tov21 cells were plated in matrigel and infected with lentiviral control or GDF15 shRNA; media and virus were changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. The percentage of anchorage-independent (AI) growth is shown. (E) Tov21 cells were plated in Boyden chambers and infected with control or GDF15 shRNA. After 24 h, the number of invaded cells was counted in ten different fields per sample. Values reflect the total number of invaded cells in triplicate cultures per group. (F) Tov21 cells were infected with lentiviral control shRNA or GDF15 shRNA for 48 h. Western blots were performed at least twice for phosphorylated and total 4EBP1, Erk1/2, p38, and Akt; representative blots are shown. Quantification is shown as a ratio of phosphorylated to total protein above each blot.

Article Snippet: Blots were probed overnight using the following antibodies: from Cell Signaling, monoclonal p-S473 Akt-XP (1:1000), and polyclonal antibodies against: total Akt (1:1000), p-Thr202/Tyr204 p42/p44 Erk1/2 (1:1000), total p42/p44 Erk1/2 (1:1000), p-Thr180/Tyr182 p38 XP (1:1000), p-Thr 37/46 4EBP1 (1:1000), total 4EBP1 (1:1000), N-Cadherin (1:1000).

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Control, Infection, shRNA, Virus, Western Blot