Journal: Scientific Reports

Article Title: Alpha-ketoglutarate promotes skeletal muscle hypertrophy and protein synthesis through Akt/mTOR signaling pathways

doi: 10.1038/srep26802

Figure Lengend Snippet: C2C12 cells were cultured for 6 d in a differentiation medium. C2C12 myotubes were then exposed to different concentrations of AKG (0, 0.5, and 2 mM) for 48 h. ( a ) Total protein levels. ( b ) Puromycin levels detected in C2C12 myotubes by Western blot. ( c ) The phosphorylation levels of mTOR, S6, 4E-BP1, eIF4E, eIF2a, and MyHC by Western blot. ( d ) IHC analysis for MHCII and phospho-S6 in C2C12 myotubes. Data are presented as mean±S.E.M. Different superscripts “a”/“b”/“c” represent significant differences between groups ( P < 0.05), and * means P < 0.05 compared with the control. β-actin served as a housekeeping gene.

Article Snippet: The PVDF membranes were then incubated with the indicated antibodies, including rabbit anti-β-actin (Bioss) and mouse puromycin antibody 12D10 (Millipore); or rabbit anti-phospho-mTOR (Ser2481) and mTOR, rabbit anti-phosphor-P70S6K (Thr389) and P70S6K1, rabbit anti-phospho-S6 (Ser235/236) and S6, rabbit anti-phospho-4E-BP1 (Thr37/46) and 4E-BP1, rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308;), rabbit anti-eIF4E, rabbit anti-phospho-eIF4E (Ser209), rabbit anti-eIF2α, rabbit anti-phospho-eIF2α (Ser251), rabbit anti-FoxO1, and rabbit anti-phospho-FoxO1 (Ser256) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Cell Culture, Western Blot, Phospho-proteomics, Control

Journal: Scientific Reports

Article Title: Alpha-ketoglutarate promotes skeletal muscle hypertrophy and protein synthesis through Akt/mTOR signaling pathways

doi: 10.1038/srep26802

Figure Lengend Snippet: ( a ) C2C12 myotubes were treated with 2 mM AKG for 1, 2, and 4 h. The expression levels of phosphorylation of mTOR, S6K, S6, 4E-BP1, eIF2a, and eIF4E were detected by Western blot. ( b ) C2C12 cells were cultured for 6 d in a differentiation medium. mTOR inhibitor rapamycin (1 μM) was used alone or co-treated with AKG (2 mM) for 48 h. Total protein levels. ( c ) The expression of puromycin determined by Western blot after C2C12 cells were co-treated with AKG and rapamycin. ( d ) The expression levels of phosphorylation of mTOR, S6, and 4E-BP1 detected by Western blot after C2C12 cells were co-treated with AKG and rapamycin. Data are presented as mean ± SEM. *means P < 0.05 compared with the control. β-actin served as a housekeeping gene control.

Article Snippet: The PVDF membranes were then incubated with the indicated antibodies, including rabbit anti-β-actin (Bioss) and mouse puromycin antibody 12D10 (Millipore); or rabbit anti-phospho-mTOR (Ser2481) and mTOR, rabbit anti-phosphor-P70S6K (Thr389) and P70S6K1, rabbit anti-phospho-S6 (Ser235/236) and S6, rabbit anti-phospho-4E-BP1 (Thr37/46) and 4E-BP1, rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308;), rabbit anti-eIF4E, rabbit anti-phospho-eIF4E (Ser209), rabbit anti-eIF2α, rabbit anti-phospho-eIF2α (Ser251), rabbit anti-FoxO1, and rabbit anti-phospho-FoxO1 (Ser256) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Phospho-proteomics, Western Blot, Cell Culture, Control

Journal: Scientific Reports

Article Title: Alpha-ketoglutarate promotes skeletal muscle hypertrophy and protein synthesis through Akt/mTOR signaling pathways

doi: 10.1038/srep26802

Figure Lengend Snippet: AKG (0.6 g/kg) and puromycin were co-injected for 3 h. Protein turnover associate protein expression was detected by Western blot. ( a ) The expression of puramycin was analyzed by Western blot. ( b ) The expression of MyHC and the phosphorylation levels of mTOR, S6, 4E-BP1, eIF4E, eIF2a in the gastrocnemius of mice. ( c ) The expression of Akt, MAFbx, and MuRF1. ( d ) The mRNA expression levels of protein turnover related genes were measured by qPCR. ( e ) The phosphorylation levels of Akt and mTOR 1 h after AKG injection detected by Western blot. Data are presented as mean ± SEM. *means P < 0.05 compared with the control. β-actin served as a housekeeping gene control.

Article Snippet: The PVDF membranes were then incubated with the indicated antibodies, including rabbit anti-β-actin (Bioss) and mouse puromycin antibody 12D10 (Millipore); or rabbit anti-phospho-mTOR (Ser2481) and mTOR, rabbit anti-phosphor-P70S6K (Thr389) and P70S6K1, rabbit anti-phospho-S6 (Ser235/236) and S6, rabbit anti-phospho-4E-BP1 (Thr37/46) and 4E-BP1, rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308;), rabbit anti-eIF4E, rabbit anti-phospho-eIF4E (Ser209), rabbit anti-eIF2α, rabbit anti-phospho-eIF2α (Ser251), rabbit anti-FoxO1, and rabbit anti-phospho-FoxO1 (Ser256) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Injection, Expressing, Western Blot, Phospho-proteomics, Control

Journal: The American journal of clinical nutrition

Article Title: Enteral delivery of proteins stimulates protein synthesis in human duodenal mucosa in the fed state through a mammalian target of rapamycin-independent pathway.

doi: 10.3945/ajcn.112.046946

Figure Lengend Snippet: FIGURE 4. Effects [medians (IQRs)] of proteins on the MNK1 pathway in duodenal mucosa. Densitometric analysis of erk 1/2 (A), MNK1 (B), and eIF4E (C) phosphorylation in the duodenal mucosa of healthy volunteers after 5 h of enteral maltodextrins alone (control) or with proteins (PP). Results (n = 12 per group) were compared by using Wilcoxon’s test. *P , 0.05 compared with the control. eIF4E, eukaryotic translation initiation factor 4E; erk, extracellular signal-regulated protein kinase; MNK1, mitogen- activated protein kinase signal-integrating kinase 1; p-, phosphorylated; PP, protein powder.

Article Snippet: An overnight incubation at 48C was done by using primary antibody:goat polyclonal antibodies antiphosphorylated eIF4E (Ser209, sc-12885; SantaCruz Biotechnology, Tebu-bio) and anti–intestinal fatty acid binding protein (iFABP) (sc-16063; SantaCruz Biotechnology), rabbit polyclonal antibodies antiphosphorylated-4E-BP1 (Ser65, sc18091-R, SantaCruz Biotechnology), antiphosphorylated phosphatidylinositol 3-kinase (PI3K) [p85 (Tyr458)/p55 (Tyr199), #4228S; Cell Signaling Technology), antiphosphorylated p27 (Thr198, sc-130603; SantaCruz Biotechnology), antiphosphorylated-MNK1 (Thr197/Thr202, 700242; Invitrogen), mouse monoclonal antibody anti–c-myc (OP10L; Merck4Biosciences), and polyclonal antibody anti-GAPDH (Sigma Aldrich).

Techniques: Phospho-proteomics, Control

Journal: Oncotarget

Article Title: Targeting Hsp27/eIF4E interaction with phenazine compound: a promising alternative for castration-resistant prostate cancer treatment

doi: 10.18632/oncotarget.20469

Figure Lengend Snippet: (a) Schematic representation of Hsp27 wild type (WT) and truncated mutant forms of Hsp27 (N1, N2, and C1). Mutants N1 and N2 correspond to two different variants of the N-terminal region of Hsp27 protein (residues 1-93, and 1-173, respectively), whereas mutant C1 corresponds to the Hsp27 C-terminal region, containing the last 113 protein residues (residues 93-205). The full-length WT Hsp27 sequence was used as control. No endogenous Hsp27 expression REG cells transiently transfected or not (NT) with plasmids containing WT Hsp27 and N1, N2 and C1 and proteins were extracted for: (b) Western Blot analysis of histidine and vinculin protein levels from total cell lysates (TCL) and (c) Western Blot analysis of histidine and eIF4E protein levels after immunoprecipitation (IP) using anti-eIF4E antibody (d) MTT quantification of REG cells viability. (e) Schematic representation of Hsp27 wild type (WT) and phosphorylation mutants of Hsp27 (3A and 3D) used in this study. In the former case, the 3A mutant of Hsp27 was specifically constructed by replacing the serine residues 15, 78 and 82 with alanine; concomitantly, the 3D Hsp27 isoforms was obtained by replacing the same serine residues with aspartic acid. (f) Western Blot analysis of histidine and vinculin protein levels from total cell lysates (TCL) and (g) Western Blot analysis of histidine and eIF4E protein levels after immunoprecipitation (IP) using anti-eIF4E antibody. (h) MTT quantification of REG cells viability was performed on cells transiently transfected with plasmids containing WT Hsp27 and phosphorylation mutants of Hsp27 prior to treatment with docetaxel in serum-free media (a condition mimicking androgen deprivation) *** P≤0.001. (i) Overall view of an equilibrated MD snapshot of the Hsp27 WT/eIF4E complex. The proteins are visualized by their van der Waals surfaces, colored as follow: Hsp27 WT, firebrick; eIF4E, khaki. The amino acids of Hsp27 mainly involved in binding eIF4E are highlighted as follows: purple, residues belonging to the WDPF domain; green, residues belonging to the flexible domain. See text for details. (j) Per residue enthalpic contribution to WT Hsp27 binding with eIF4E. Only those Hsp27 amino acids affording a meaningful contribution to protein-protein formation contributing (ΔH bind,res < -0.80 kcal/mol) are shown for clarity. From this analysis, it further appears that the Hsp27 α-crystallin domain is practically ineffective in Hsp27/eIF4E binding since residues belonging to this Hsp27 region display a negligible contribution to protein-protein binding enthalpy. (k) Overall view of equilibrated MD snapshots of the eIF4E in complex with Hsp27 N1, N2 and C1 truncated mutant isoforms. In each panel, the proteins are visualized by their van der Waals surfaces, colored as follows: Hsp27s, firebrick; eIF4E, khaki. The amino acids of Hsp27 mainly involved in binding eIF4E are highlighted as follows: purple, residues belonging to the WDPF domain; green, residues belonging to the flexible domain. Interestingly, the overall structure of Hsp27 N1 is de facto strongly affected by the loss of the α-crystallin domain, which plays a major role in the overall correct chaperon folding required for effective binding to eIF4E.

Article Snippet: Western blot (WB) was performed with 1/3000 mouse anti-polyHistidine antibody (Sigma-Aldrich, USA), 1/1000 rabbit anti-eIF4E antibody (Cell Signaling, Ozyme), 1/2500 anti-mouse IgG HRP conjugate antibody (Promega, France), 1/1000 anti-rabbit Trueblot IgG HRP conjugate antibody and 1/1000 anti-mouse Trueblot IgG HRP conjugate antibody (eBiosciences), 1/5000 anti-rabbit IgG HRP conjugate antibody (Santa Cruz Biotechnology, Germany), 1/5000 rabbit Hsp27 antibody (Assay Designs, FranceLoading levels were normalized using 1/2000 mouse anti-vinculin antibody (Sigma-Aldrich).

Techniques: Mutagenesis, Sequencing, Expressing, Transfection, Western Blot, Immunoprecipitation, Construct, Binding Assay, Protein Binding

Journal: Oncotarget

Article Title: Targeting Hsp27/eIF4E interaction with phenazine compound: a promising alternative for castration-resistant prostate cancer treatment

doi: 10.18632/oncotarget.20469

Figure Lengend Snippet: Binding free energies (ΔG bind ) and binding free energy differences (ΔΔG bind ) for the Hsp27 WT, N1, N2 and C2 truncated mutants in complex with eIF4E. (ΔΔG bind =ΔG bind(WT) - ΔG bind(truncated) )

Article Snippet: Western blot (WB) was performed with 1/3000 mouse anti-polyHistidine antibody (Sigma-Aldrich, USA), 1/1000 rabbit anti-eIF4E antibody (Cell Signaling, Ozyme), 1/2500 anti-mouse IgG HRP conjugate antibody (Promega, France), 1/1000 anti-rabbit Trueblot IgG HRP conjugate antibody and 1/1000 anti-mouse Trueblot IgG HRP conjugate antibody (eBiosciences), 1/5000 anti-rabbit IgG HRP conjugate antibody (Santa Cruz Biotechnology, Germany), 1/5000 rabbit Hsp27 antibody (Assay Designs, FranceLoading levels were normalized using 1/2000 mouse anti-vinculin antibody (Sigma-Aldrich).

Techniques: Binding Assay

Journal: Oncotarget

Article Title: Targeting Hsp27/eIF4E interaction with phenazine compound: a promising alternative for castration-resistant prostate cancer treatment

doi: 10.18632/oncotarget.20469

Figure Lengend Snippet: (a) To by-pass technical parameters that could prevent us from measuring a BRET signal (presence and/or position of the reporter proteins that could disrupt the Hsp27/eIF4E interaction, physical distance between Rluc and YFP), the interaction between these two proteins with all possible couple combinations (n=8) of plasmids were tested. These couples of plasmids were transfected separately in HEK293T cells at different concentrations in order to find the ideal ratios and obtain the BRET signal. For the construction of BRET plasmids, we merged each gene (Hsp27 and eIF4E) with Rluc or YFP in N-terminal (N-ter) or C-terminal (C-ter) part. After, we tested the interaction between these two proteins with all the possible couple combinations of plasmids, on BRET on living cells or cells extracts. (b) Hsp27/eIF4E interaction in BRET experiment was investigated in HEK293T cell extracts. The principle was similar to BRET assay in living cells; except that the plasmids were transfected separately in HEK293T cells and that the couple combinations were tested in vitro by mixing proteins extracted from these transfected cells. HEK293T cells were co-transfected with 0.2 μg of BRET donor plasmid N-ter luc/eIF4E, and 0 to 1 μg of BRET acceptor plasmid Hsp27/YFP C-ter . The empty vector (pEYFP-C) was used to equalize DNA amounts to 1.2 μg in each sample. The reading of optic density was performed after the addition of coelenterazine in order to obtain the BRET signal. (c) HEK293T cells were transfected separately with a BRET donor plasmid N-ter luc/eIF4E or BRET acceptor plasmid Hsp27/YFP C-ter . Δ=(YFP/Luc-YFP 0 /Luc 0 )*1000. (d) Derivatives of phenazines that have been described to have an anti-tumor activity as well as a structure similar to inhibitors of eIF4E/eIF4G interaction. (e) Compound 4E2RCat, which is described in literature to be an inhibitor of the eIF4E/eIF4G interaction. (f) HEK293T cells were transfected separately with a BRET donor plasmid N-ter luc/eIF4E or BRET acceptor plasmid Hsp27/YFP C-ter . Total proteins were extracted from cells and used for BRET assay: 1 μg of lysate containing a BRET donor and 0 to 15 μg of lysate containing a BRET acceptor were pre-incubated separately with different concentrations (0, 20, 50 and 100μM; green, yellow, orange and red lines respectively) of compound 14 during 2h. As control experiment, cell extracts were pre-incubated with DMSO alone, at the higher concentration (1%) we used to dilute compound (control DMSO, blue line). Donor and acceptor were mixed for 30 min and the reading of optic density was performed after the addition of coelenterazine in order to obtain the BRET signal. (g) The same experiment was performed with another couple of protein: CCND3/luc and CDK6/YFP (4μg of lysate containing BRET donor was used). Δ=(YFP/Luc-YFP 0 /Luc 0 )*1000. (h) PC-3 cells were treated at 100μM with compound 14 (right panel, Bar=10 μm) and DMSO (left panel, Bar = 20μm) as control. Auto-fluorescence of compound 14 (green) and staining of the nucleus by DAPI (blue) was observed. PC-3 cells were treated with DMSO (control) or compound 14 during 48h and proteins were extracted for: (i) Western Blot analysis of Hsp27, eIF4E protein levels after immunoprecipitation (IP) using eIF4E rabbit antibody or IgG rabbit (control) (j) Western Blot analysis of Hsp27, eIF4E, Vinculin and protein levels from total protein extracts.

Article Snippet: Western blot (WB) was performed with 1/3000 mouse anti-polyHistidine antibody (Sigma-Aldrich, USA), 1/1000 rabbit anti-eIF4E antibody (Cell Signaling, Ozyme), 1/2500 anti-mouse IgG HRP conjugate antibody (Promega, France), 1/1000 anti-rabbit Trueblot IgG HRP conjugate antibody and 1/1000 anti-mouse Trueblot IgG HRP conjugate antibody (eBiosciences), 1/5000 anti-rabbit IgG HRP conjugate antibody (Santa Cruz Biotechnology, Germany), 1/5000 rabbit Hsp27 antibody (Assay Designs, FranceLoading levels were normalized using 1/2000 mouse anti-vinculin antibody (Sigma-Aldrich).

Techniques: Transfection, Bioluminescence Resonance Energy Transfer, In Vitro, Plasmid Preparation, Activity Assay, Incubation, Concentration Assay, Fluorescence, Staining, Western Blot, Immunoprecipitation

Journal: Oncotarget

Article Title: Targeting Hsp27/eIF4E interaction with phenazine compound: a promising alternative for castration-resistant prostate cancer treatment

doi: 10.18632/oncotarget.20469

Figure Lengend Snippet: (a) (Left panel) Zoomed view of an MD equilibrated snapshot of the 14 in complex with eIF4E. Specifically, the ligand is docked between a α-helix of eIF4E protein spanning residue L117*-E132* and a structurally hybrid region from E185* to I193*. The ligand is portrayed in ball-and-stick representation and colored by element (C, gray; N, blue; O, red). The main protein residues involved in compound binding are depicted as gold sticks and labelled. Transparent light blue spheres represent water oxygen atoms, while chlorine and sodium ions and counterions are shown as green and purple spheres, respectively. Hydrogen atoms are omitted for clarity. (Right Panel) Per residue binding enthalpy decomposition for eIF4E residues mainly involved in binding with 14. Only those Hsp27 amino acids affording a meaningful contribution to protein-drug formation (ΔH bind,res* < -0.50 kcal/mol) are shown for clarity. The network of stabilizing hydrophobic interaction involves the two C 12 alkyl chains of the 14 and the side chain of the protein residue L117*, N118*, F129*, L189*, P190*, K192*, and I193*. The two amine substituents of 14 are engaged in two persistent polar interactions with the carboxylic side chain of E132* and E185*. In addition, a further stabilizing interaction via a weak hydrogen bond between the side chain of R128* and a nitrogen atom of the phenazine ring is detected. (b) (Left panel) MD simulation distance between the charged side chains of E132* and R186* in the eIF4E alone (red line) and in the eIF4E/14 complex (salmon line). In the eIF4E free protein, the two α-helixes spanning residues Q122*-I138* and R173*-G188*, respectively, are persistently stabilized by the presence of permanent interaction points between the side chain of their residues. In particular, a strong polar interaction between the charged side chain of the amino acids E132* and R186* is detected. As shown, the Average Dynamics Length (ADL) of this interaction between the two involved atoms is 2.96 Å, and its persistence is verified along the entire MD run. Conversely, the same distance progressively increases during the first part of the simulations and finally settles around 9Å when the eIF4F is simulated in presence of the ligand. The reason for this behavior can be explained by that fact that E132* is engaged in a polar interaction with 14, as demonstrated by the complementary trend exhibited of the corresponding distance between the carboxylic moiety of E132* and the amine group of 14 shown in the right panel (MD simulation distance between the N2 nitrogen atom of 14 and the charged side chain of E132* (blue line), and between the N3 nitrogen atom of 14 and the charged side chain of E185* (green line). (c) (Left panel) Overall view of an equilibrated MD snapshot of the Hsp27 WT/eIF4E/14 complex. All molecules are visualized by their van der Waals surfaces, colored as follow: WT Hsp27, firebrick; eIF4E, khaki; 14, navy blue. The amino acids of Hsp27 mainly involved in binding eIF4E are highlighted as follows: purple, residues belonging to the WDPF domain; green, residues belonging to the flexible domain. The residues of eIF4E mainly involved in binding with 14 are depicted in gold. (Upper right panel) Binding free energies (ΔG bind ) and binding free energy differences (ΔG bind ) for the WT Hsp27/eIF4E (plain filled columns) and the WT Hsp27/eIF4E/14 (patterned filled columns) complexes. (Lower right panel) Comparison of the clustered per residue enthalpic contribution to binding for WT Hsp27/eIF4E and WT Hsp27/eIF4E/14 complexes. (d) ITC experiment of 14/eIF4E binding: (left) raw data; (middle) titration curve; (right) binding thermodynamics parameters.

Article Snippet: Western blot (WB) was performed with 1/3000 mouse anti-polyHistidine antibody (Sigma-Aldrich, USA), 1/1000 rabbit anti-eIF4E antibody (Cell Signaling, Ozyme), 1/2500 anti-mouse IgG HRP conjugate antibody (Promega, France), 1/1000 anti-rabbit Trueblot IgG HRP conjugate antibody and 1/1000 anti-mouse Trueblot IgG HRP conjugate antibody (eBiosciences), 1/5000 anti-rabbit IgG HRP conjugate antibody (Santa Cruz Biotechnology, Germany), 1/5000 rabbit Hsp27 antibody (Assay Designs, FranceLoading levels were normalized using 1/2000 mouse anti-vinculin antibody (Sigma-Aldrich).

Techniques: Binding Assay, Titration

Journal: Cell reports

Article Title: Revealing molecular pathways for cancer cell fitness through a genetic screen of the cancer translatome

doi: 10.1016/j.celrep.2021.109321

Figure Lengend Snippet:

Article Snippet: p-eIF4E Ser209 , Cell Signaling Technology , Cat# 9721; RRID:AB_561095.

Techniques: Virus, Recombinant, Activation Assay, Extraction, Reverse Transcription, SYBR Green Assay, Reporter Assay, RNA Sequencing, Expressing, CRISPR, Software

Journal: PLoS ONE

Article Title: Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells

doi: 10.1371/journal.pone.0066189

Figure Lengend Snippet: ( A–B ) S1P increases HIF-1α expression in a time- and concentration dependent manner. Cells were stimulated with 100 nM S1P for the indicated times or with the indicated concentrations for 6 h. A lysate of CoCl 2 -treated cells was used as positive control for HIF-1α (+). ( C ) Hypoxia increases HIF-1α protein expression. Cells were incubated in hypoxia (1% O 2 ) for the indicated times. ( D ) S1P increases expression from promoters containing the HRE sequence. ML-1 cells were transfected with a HRE-Luc or a negative control TK-Luc plasmid and treated with S1P (100 nM) or CoCl 2 (150 µM) for 7 h. ( E ) S1P increases expression of HIF-1 target genes in a HIF-1α-dependent manner. ML-1 cells were transfected with control siRNA (siC) or siRNA against HIF-1α (siHIF) and treated with S1P (100 nM) or incubated in hypoxia (H, 1% O 2 ) for 9 h. Hypoxia-treated samples were used as a positive control for HIF-1 target gene expression. Results are mean ± SEM, n ≥ 3. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate statistically significant difference between S1P, COCl 2 or hypoxia treatment and respective vehicle or siRNA control.

Article Snippet: Antibodies for β-actin, VEGFR-2, HIF-1α (WB), hydroxy-HIF-1α (Pro564), phospho-eIF-4E (Ser209), eIF-4E, phospho-4E-BP1 (Ser65) and 4E-BP1 as well as horseradish peroxidase-conjugated anti-rat antibody were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Concentration Assay, Positive Control, Incubation, Sequencing, Transfection, Negative Control, Plasmid Preparation, Control, Targeted Gene Expression

Journal: PLoS ONE

Article Title: Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells

doi: 10.1371/journal.pone.0066189

Figure Lengend Snippet: Inhibition of ( A ) G i proteins, ( B ) S1P 3 or ( C ) S1P 1 and S1P 3 and ( D ) knockdown of S1P 3 prevents S1P-induced HIF-1α expression. Cells were pretreated with Pertussis toxin (Ptx, 100 ng/ml, 24 h), CAY10444 (CAY, 10 µM, 1 h) or VPC-23019 (VPC, 10 µM, 30 min) or transfected with control siRNA (siC) or S1P receptor siRNA (si1-3) and stimulated with S1P (100 nM) for 6 h. Results are mean ± SEM, n ≥ 3. *P < 0.05 and ***P < 0.001 indicate statistically significant difference between S1P treatment and respective vehicle or siRNA control, ooo P < 0.001 indicates statistically significant difference between inhibitor treatment and vehicle control.

Article Snippet: Antibodies for β-actin, VEGFR-2, HIF-1α (WB), hydroxy-HIF-1α (Pro564), phospho-eIF-4E (Ser209), eIF-4E, phospho-4E-BP1 (Ser65) and 4E-BP1 as well as horseradish peroxidase-conjugated anti-rat antibody were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Knockdown, Expressing, Transfection, Control

Journal: PLoS ONE

Article Title: Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells

doi: 10.1371/journal.pone.0066189

Figure Lengend Snippet: Inhibition of ( A ) PI3K, ( B ) mTOR and ( C ) MEK prevents S1P-induced HIF-1α expression. Cells were preincubated with wortmannin (W, 10 µM, 30 min), rapamycin (Rapa, 100 ng/ml, 1h) or with U0126 (U0, 10 µM, 1 h) and stimulated with S1P (100 nM) for 6 h. ( D ) Phorbol 12-myristate 13-acetate (PMA) induces HIF-1α expression. Cells were stimulated with PMA (100 nM) for the indicated times. ( E ) PKCβI mediates S1P-induced HIF-1α expression transfected with PKC isoform specific siRNA and stimulated with S1P (100 nM) for 6 h. Results are mean ± SEM, n ≥ 3. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate statistically significant difference between S1P treatment and respective vehicle or siRNA control.

Article Snippet: Antibodies for β-actin, VEGFR-2, HIF-1α (WB), hydroxy-HIF-1α (Pro564), phospho-eIF-4E (Ser209), eIF-4E, phospho-4E-BP1 (Ser65) and 4E-BP1 as well as horseradish peroxidase-conjugated anti-rat antibody were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Expressing, Transfection, Control

Journal: PLoS ONE

Article Title: Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells

doi: 10.1371/journal.pone.0066189

Figure Lengend Snippet: ( A ) S1P prolongs HIF-1α half-life. Cells were either left untreated, treated with S1P (100 nM) for 6 h, incubated in hypoxia (1% O 2 ) for 6 h or treated with CoCl 2 (150 µM) for 3 h before the cycloheximide chase (Chx, 5 µg/ml). S1P, hypoxic conditions or CoCl 2 were present throughout the chase. Time points are mean ± SEM, n = 3–10. Curve fit was done with the one phase exponential decay equation. ( B ) S1P does not inhibit binding of pVHL to HIF-1α. Cells were treated with S1P (100 nM) for 6 h. The level of co-immunoprecipitated HIF-1α was compared with the level of immunoprecipitated pVHL and IgG bands were used as a loading control. **P < 0.01 indicates statistically significant difference between S1P treatment and vehicle control.

Article Snippet: Antibodies for β-actin, VEGFR-2, HIF-1α (WB), hydroxy-HIF-1α (Pro564), phospho-eIF-4E (Ser209), eIF-4E, phospho-4E-BP1 (Ser65) and 4E-BP1 as well as horseradish peroxidase-conjugated anti-rat antibody were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Incubation, Binding Assay, Immunoprecipitation, Control

Journal: PLoS ONE

Article Title: Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells

doi: 10.1371/journal.pone.0066189

Figure Lengend Snippet: ( A–D ) S1P induces phosphorylation of mTOR, eIF-4E and 4E-BP1 via PI3K and MEK and phosphorylation of p70S6K via PI3K. Cells were preincubated with wortmannin (W, 10 µM, 30 min) or U0126 (U0, 10 µM, 1 h) and stimulated with S1P (100 nM) for 30 min. ( E–G ) S1P induces phosphorylation of eIF-4E, 4E-BP1 and p70S6K via mTOR. Cells were preincubated with rapamycin (Rapa, 100 ng/ml, 1 h) and stimulated with S1P (100 nM) for 30 min. ( H ) Inhibition of p70S6K prevents S1P-induced HIF-1α expression. Cells were preincubated with p70S6K inhibitor (p70i, 10 µM, 1 h) and stimulated with S1P (100 nM) for 6 h. Results are mean ± SEM, n ≥ 3. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate statistically significant difference between S1P treatment and respective vehicle control, oo P < 0.01 and ooo P < 0.001 indicate statistically significant difference between inhibitor treatment and vehicle control.

Article Snippet: Antibodies for β-actin, VEGFR-2, HIF-1α (WB), hydroxy-HIF-1α (Pro564), phospho-eIF-4E (Ser209), eIF-4E, phospho-4E-BP1 (Ser65) and 4E-BP1 as well as horseradish peroxidase-conjugated anti-rat antibody were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Inhibition, Expressing, Control

Journal: PLoS ONE

Article Title: Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells

doi: 10.1371/journal.pone.0066189

Figure Lengend Snippet: ( A ) Inhibition of HIF-1 attenuates S1P-induced migration. Cells were preincubated with HIF-1 inhibitor (HIFi, 10 µM, 30 min) and S1P (100 nM, 30 min) and allowed to migrate towards serum for 8 h. ( B ) Down-regulation of HIF-1α decreases basal migration. Cells were transfected with HIF-1α siRNA and allowed to migrate towards serum and S1P (100 nM) for 8 h. ( C ) Down-regulation of HIF-1α attenuates S1P-induced migration. Cells were transfected with HIF-1α siRNA and allowed to migrate towards S1P (100 nM) for 20 h. ( D ) Inhibition of p70S6K decreases basal migration and prevents S1P-induced migration. Cells were preincubated with p70S6K inhibitor (p70i, 10 µM, 30 min) and S1P (100 nM, 30 min) and allowed to migrate towards serum for 8 h. ( E ) Down-regulation of S1P 3 attenuates S1P-induced migration. Cells were transfected with S1P 3 siRNA and allowed to migrate towards serum and S1P (100 nM) for 8 h. Results are mean ± SEM, n ≥ 3. *P < 0.05 and ***P < 0.001 indicate statistically significant difference between S1P treatment and respective vehicle or siRNA control, o P < 0.05 and ooo P < 0.001 indicate statistically significant difference between siRNA treatment and control siRNA, between siRNA+S1P treatment and control siRNA+S1P or between inhibitor treatment and vehicle control.

Article Snippet: Antibodies for β-actin, VEGFR-2, HIF-1α (WB), hydroxy-HIF-1α (Pro564), phospho-eIF-4E (Ser209), eIF-4E, phospho-4E-BP1 (Ser65) and 4E-BP1 as well as horseradish peroxidase-conjugated anti-rat antibody were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Migration, Transfection, Control

Journal: PLoS ONE

Article Title: Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells

doi: 10.1371/journal.pone.0066189

Figure Lengend Snippet: S1P stimulation up-regulates the HIF-1α protein in normoxia. This effect is dependent on activity of S1P 3 and G i as well as their downstream effectors PKCβI, PI3K and MEK (S1P receptor signaling reviewed in 3 and 4). We suggest S1P to regulate both stability and translation of HIF-1α. S1P stimulation increases phosphorylation of mTOR via MEK and PI3K and phoshorylation of p70S6K, eIF-4E and 4E-BP1 via MEK and/or PI3K/mTOR and inhibition of p70S6K prevents S1P-induced up-regulation of HIF-1α. HIF-1 is involved in both basal and S1P-induced ML-1 migration.

Article Snippet: Antibodies for β-actin, VEGFR-2, HIF-1α (WB), hydroxy-HIF-1α (Pro564), phospho-eIF-4E (Ser209), eIF-4E, phospho-4E-BP1 (Ser65) and 4E-BP1 as well as horseradish peroxidase-conjugated anti-rat antibody were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay, Phospho-proteomics, Inhibition, Migration