Journal:

Article Title: Translocation of FGF-1 and FGF-2 across Vesicular Membranes Occurs during G 1 -Phase by a Common Mechanism

doi: 10.1091/mbc.E03-08-0589

Figure Lengend Snippet: In vitro farnesylation and biological activity of FGF-2-CaaX. (A) Recombinant FGF-2 or FGF-2-CaaX were incubated in a reticulocyte lysate system in the presence of [3H]farnesyl pyrophosphate in the absence or presence of B581. Samples were treated with heparin-Sepharose (lanes 1-3) or subjected to immunoprecipitation using anti-FGF-2 antibodies adsorbed to protein A-Sepharose (lanes 4-6) and analyzed by SDS-PAGE and fluorography. The arrow indicates the migration of farnesylated FGF-2-CaaX. (B) Serum-starved NIH/3T3 cells were treated with heparin and the indicated amount of FGF-2 or FGF-2-CaaX for 10 min. The cells were lysed in SDS sample buffer, sonicated, and analyzed by SDS-PAGE and Western blotting using antibodies against total p44/42 MAP kinase (bottom panel). The membrane was stripped and reprobed using antibodies against the phosphorylated form of p44/42 MAP kinase (top panel). (C) Serum-starved NIH/3T3 cells were treated with heparin and increasing amounts of FGF-2 or FGF-2-CaaX for 24 h. During the last 6 h of incubation, 1 μCi/ml [3H]thymidine was present in the medium. The cells were extracted with 5% TCA and the radioactivity incorporated into TCA-insoluble material was measured. (D) FGF-2 or FGF-2-CaaX conjugated to Alexa 488 maleimide was added to NIH/3T3 cells transfected with pcDNA3 vector encoding FGFR1 and incubated for 15 min at 37°C. The cells were then fixed, permeabilized, and treated with a mouse antibody against EEA1. The cells were further treated with Cy3-conjugated anti-mouse antibody. The arrows point to examples of structures positive for EEA1 and FGF-2 or FGF-2-CaaX.

Article Snippet: Rabbit polyclonal anti FGF-2 was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vitro, Activity Assay, Recombinant, Incubation, Immunoprecipitation, SDS Page, Migration, Sonication, Western Blot, Radioactivity, Transfection, Plasmid Preparation

Journal:

Article Title: Translocation of FGF-1 and FGF-2 across Vesicular Membranes Occurs during G 1 -Phase by a Common Mechanism

doi: 10.1091/mbc.E03-08-0589

Figure Lengend Snippet: In vivo prenylation of FGF-2-CaaX in NIH/3T3 cells. (A) After 24 h serum-starved cells were preincubated for 2 h with [14C]mevalonolactone and 1 μg/ml lovastatin and then treated for 6 h with heparin and either FGF-2 (lane 1) or FGF-2-CaaX (lane 2). The cells were lysed and the cellular material was adsorbed onto heparin-Sepharose and analyzed by SDS-PAGE and fluorography. The arrows indicate the migration of in vivo prenylated and in vitro farnesylated FGF-2-CaaX. In vitro farnesylated FGF-2-CaaX (lane 3) is shown as marker. (B) Serum-starved cells were incubated for 6 h with heparin and ∼10 ng/ml [35S]methionine-labeled 18-kDa FGF-2. Cells were washed with 2 M NaCl in Na-acetate buffer, pH 4.0, and lysed. The material present in the high-salt/low-pH wash (lane 1), and the cellular material (lane 2) was adsorbed onto heparin-Sepharose and analyzed by SDS-PAGE and fluorography. [35S]methionine-labeled 16-kDa form of FGF-2 (lane 3) is shown as marker. (C) Cells were pretreated as in A and then incubated for 6 h with the indicated amounts of FGF-2-CaaX in the absence (lanes 1-3) or presence (lanes 4-6) of heparin. After lysis, cellular material was analyzed as in A.

Article Snippet: Rabbit polyclonal anti FGF-2 was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vivo, SDS Page, Migration, In Vitro, Marker, Incubation, Labeling, Lysis

Journal:

Article Title: Translocation of FGF-1 and FGF-2 across Vesicular Membranes Occurs during G 1 -Phase by a Common Mechanism

doi: 10.1091/mbc.E03-08-0589

Figure Lengend Snippet: Effect of inhibitors of PI-3 kinase on the prenylation of FGF-2-CaaX in vivo. (A) CPAE cells were pretreated as in Figure 2A and then treated for 6 h with heparin and either FGF-2 (lane 1) or FGF-2-CaaX (lanes 2-4). The incubation was carried out in the absence (lanes 1 and 2) or presence of 50 μM LY294002 (lane 3), or 200 nM wortmannin (lane 4). After lysis, cellular material was analyzed as in Figure 2A. (B) NIH/3T3 cells were pretreated as in Figure 2A and then treated for 6 h with heparin and FGF-2-CaaX. The incubation was carried out in the absence (lane 1) or presence of 50 μM LY294002 (lane 2) or 150 nM wortmannin (lane 3). After lysis, cellular material was analyzed as in Figure 2A.

Article Snippet: Rabbit polyclonal anti FGF-2 was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vivo, Incubation, Lysis

Journal:

Article Title: Translocation of FGF-1 and FGF-2 across Vesicular Membranes Occurs during G 1 -Phase by a Common Mechanism

doi: 10.1091/mbc.E03-08-0589

Figure Lengend Snippet: In vivo prenylation of FGF-2-CaaX is not blocked by treatment with brefeldin A, nocodazole and cytochalasin D. (A) NIH/3T3 cells were treated without or with 2 μg/ml brefeldin A for 30 min. The Golgi apparatus was visualized with anti-β-COP and Cy3-conjugated anti-rabbit antibody using confocal microscopy. (B) NIH/3T3 cells were treated without or with 10 μg/ml cytochalasin D for 30 min. Actin was visualized with rhodamine-conjugated phalloidin. (C) NIH/3T3 cells were treated without or with 33 μM nocodazole for 30 min. Microtubuli were visualized with antitubulin and rhodamine-conjugated anti-mouse antibody. (D) NIH/3T3 cells were pretreated as in Figure 2A and then treated for 6 h with heparin and FGF-2-CaaX in the absence (lane 1) or presence of 2 μg/ml brefeldin A (lane 2), 10 μg/ml cytochalasin D (lane 3), or 33 μM nocodazole (lane 4). After lysis, cellular material was analyzed as in Figure 2A.

Article Snippet: Rabbit polyclonal anti FGF-2 was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vivo, Confocal Microscopy, Lysis

Journal:

Article Title: Translocation of FGF-1 and FGF-2 across Vesicular Membranes Occurs during G 1 -Phase by a Common Mechanism

doi: 10.1091/mbc.E03-08-0589

Figure Lengend Snippet: Effect of various inhibitors of lumenal acidification of intracellular vesicles on the ability of FGF-2-CaaX to become prenylated in vivo. (A) NIH/3T3 cells were pretreated as in Figure 2A and then treated for 6 h with heparin and FGF-2-CaaX in the absence (lane 1) or presence of 1 μM monensin (lane 2), 10 nM bafilomycin A1 (lane 3), 20 mM NH4Cl (lane 4), or 100 μM chloroquine (lane 5). After lysis, cellular material was analyzed as in Figure 2A. (B) NIH/3T3 cells were pretreated as in Figure 2A and then treated for 6 h with heparin and FGF-2-CaaX in the absence (lane 1) or the presence of increasing concentrations of concanamycin A (lanes 2-4). After lysis, cellular material was analyzed as in Figure 2A. (C) FGF-2 conjugated to Cy3 maleimide and FGF-2-CaaX conjugated to Alexa 488 maleimide were added to NIH/3T3 cells overexpressing FGFR1 and incubated for 2 h at 37°C. In some cases, the cells were pretreated with 10 nM bafilomycin A. The cells were then fixed and analyzed by confocal microscopy.

Article Snippet: Rabbit polyclonal anti FGF-2 was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vivo, Lysis, Incubation, Confocal Microscopy

Journal:

Article Title: Translocation of FGF-1 and FGF-2 across Vesicular Membranes Occurs during G 1 -Phase by a Common Mechanism

doi: 10.1091/mbc.E03-08-0589

Figure Lengend Snippet: Effect of depolarization and repolarization of the membrane of intracellular vesicles on the ability of FGF-2-CaaX to become prenylated in vivo. (A) H+ are pumped from the cytosol into the lumen of the vesicles by V-type H+-ATPase (red) lowering lumenal pH and creating a membrane potential (positive inside the vesicle). The membrane potential is partly compensated for by the influx of Cl- counterions (not indicated). Inhibition of vacuolar proton pumps by bafilomycin A1 or concanamycin A causes depolarization of the membrane. Monensin or nigericin (green) exchange H+ accumulated in the vesicle lumen for K+ present in the cytosol, which raises the lumenal pH but does not dissipate the membrane potential. When both monensin (or nigericin) and valinomycin (blue) are present, efflux of K+ from the lumen to the cytosol results in the dissipation of the membrane potential. Endosomes also contain Na+/K+-ATPase (yellow), which is usually inactive because of deficiency in lumenal K+. In the presence of monensin (or nigericin) lumenal K+ can be replenished (in exchange for Na+) leading to reactivation of the Na+/K+-ATPase and regeneration of the membrane potential even when V-type H+-ATPase is blocked. Under these conditions, treatment with ouabain (inhibitor of Na+/K+-ATPase) will lead again to depolarization of the membrane. (B) CPAE cells were pretreated as in Figure 2A and then treated for 6 h with heparin and FGF-2-CaaX in the absence (lane 1) or presence of valinomycin (lane 2), monensin (lane 3), nigericin (lane 5), or a combination of valinomycin and either monensin (lane 4) or nigericin (lane 6). After lysis, cellular material was analyzed as in Figure 2A. (C) NIH/3T3 cells were pretreated as in Figure 2A and then treated for 6 h with heparin and FGF-2-CaaX in the absence (lane 1) or presence of monensin (lane 2), ouabain (lane 3), bafilomycin A1 (lane 4), or a combination of both bafilomycin A1 and either monensin (lane 5) or monensin and ouabain (lane 6). After lysis, cellular material was analyzed as in Figure 2A. (D) CPAE cells were pretreated as in Figure 2A and then treated for 6 h with heparin and FGF-2-CaaX in the absence (lane 1) or presence of monensin (lane 2), bafilomycin A1 (lane 3), or a combination of both monensin and bafilomycin A1 (lane 4). After lysis, cellular material was analyzed as in Figure 2A.

Article Snippet: Rabbit polyclonal anti FGF-2 was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vivo, Inhibition, Lysis

Journal:

Article Title: Translocation of FGF-1 and FGF-2 across Vesicular Membranes Occurs during G 1 -Phase by a Common Mechanism

doi: 10.1091/mbc.E03-08-0589

Figure Lengend Snippet: Effect of various inhibitors and their combinations on the ability of FGF-2 to stimulate phosphorylation of MAP-kinase in NIH/3T3 cells. Serum-starved cells were preincubated for 2 h in the absence (lanes 1 and 2) or presence of 50 μM LY294002 (lane 3), 10 nM bafilomycin A1 (lane 4), or a combination of 1 μM monensin and either 1 μM valinomycin (lane 5) or 10 nM bafilomycin A1 and 100 μM ouabain (lane 6). The cells were left untreated (lane 1) or treated for 3 h with heparin and 5 ng/ml FGF-2 (lanes 2-6). The cells were subsequently lysed and analyzed by Western blotting with anti-p44/42 MAP-kinase antibodies (bottom panel). The membrane was stripped and reprobed with antiphosphorylated-p44/42 MAP-kinase antibodies (top panel).

Article Snippet: Rabbit polyclonal anti FGF-2 was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot

Journal:

Article Title: Translocation of FGF-1 and FGF-2 across Vesicular Membranes Occurs during G 1 -Phase by a Common Mechanism

doi: 10.1091/mbc.E03-08-0589

Figure Lengend Snippet: Dependence of FGF-1 and FGF-2 translocation on the cell cycle in NIH/3T3 cells. (A) Cells were serum-starved for the indicated period of time and then preincubated for 2 h with [14C]mevalonolactone and 1 μg/ml lovastatin. Next, the cells were treated for 6 h with heparin and either: FGF-1 and FGF-1-CaaX (top panel), or FGF-2 and FGF-2-CaaX (bottom panel). After lysis, cellular material was analyzed as in Figure 2A. (B) Cells were pretreated as in Figure 2A. Next, the cells were treated for the indicated period of time with heparin and either: FGF-1-CaaX (top panel) or FGF-2-CaaX (bottom panel). After lysis, cellular material was analyzed as in Figure 2A. (C) Cells were serum-starved for 24 h and treated with heparin and 10 ng/ml either: FGF-1 (•) or FGF-2 (○) for the indicated period of time. During the last 1 h of incubation 1 μCi/ml [3H]thymidine was present in the medium. Next, cells were precipitated with 5% TCA and the incorporated radioactivity was measured. For comparison the same graph shows the plot of band intensities from B (in arbitrary units), which represent prenylated FGF-1-CaaX (▪) or prenylated FGF-2-CaaX (□).

Article Snippet: Rabbit polyclonal anti FGF-2 was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Translocation Assay, Lysis, Incubation, Radioactivity

Journal:

Article Title: Translocation of FGF-1 and FGF-2 across Vesicular Membranes Occurs during G 1 -Phase by a Common Mechanism

doi: 10.1091/mbc.E03-08-0589

Figure Lengend Snippet: FGF-2 translocation is induced by FGF-1 and FGF-2 treatment of NIH/3T3 cells. (A) Schematic representation of the time-course of the experiment. (B) Serum starved cells were preincubated with [14C]mevalonolactone, 1 μg/ml lovastatin and heparin. In one case 100 ng/ml FGF-2-CaaX was added for the entire 6 h incubation (lane 1). In other cases, the cells were for the initial 4 h left untreated (lane 2) or were treated with 10% serum (lane 3), 5 ng/ml FGF-2 (lane 4) or 5 ng/ml FGF-1 (lane 5) and then 100 ng/ml FGF-2-CaaX was added for the final 2 h of incubation. After lysis, cellular material was analyzed as in Figure 2A. (C) NIH/3T3 cells were pretreated as in Figure 2A and then treated for 6 h with heparin and either FGF-1 or FGF-1-CaaX in the absence (lanes 1 and 2) or presence of cycloheximide (lane 3). After lysis, cellular material was analyzed as in Figure 2A.

Article Snippet: Rabbit polyclonal anti FGF-2 was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Translocation Assay, Incubation, Lysis

Journal: Theranostics

Article Title: Enhanced antitumor efficacy of biocompatible magnetosomes for the magnetic hyperthermia treatment of glioblastoma

doi: 10.7150/thno.18927

Figure Lengend Snippet: (a), H&E and (b), Perls Prussian blue section of a subcutaneous GL-261 tumor (blue circles) injected with 25 µg in iron of M-PLL per mm 3 of tumor and collected 6 hours after the first MS (M-PLL (6H) + AMF). (c), enlargement of (b).

Article Snippet: The GL-261 mouse glioma cell line (ATCC, USA) was cultivated in RPMI 1640 medium (GE Healthcare HyClone™, USA), supplemented with 10 % FBS at 37 °C in humidified atmosphere containing 5 % CO 2 .

Techniques: Injection

Journal: Theranostics

Article Title: Enhanced antitumor efficacy of biocompatible magnetosomes for the magnetic hyperthermia treatment of glioblastoma

doi: 10.7150/thno.18927

Figure Lengend Snippet: (a), H&E and (b), Perls Prussian blue section of a subcutaneous GL-261 tumor (blue circles) injected with 25 µg in iron of IONPs per mm 3 of tumor and collected 6 hours after the first MS (IONPs (6H) + AMF). (c), enlargement of (b).

Article Snippet: The GL-261 mouse glioma cell line (ATCC, USA) was cultivated in RPMI 1640 medium (GE Healthcare HyClone™, USA), supplemented with 10 % FBS at 37 °C in humidified atmosphere containing 5 % CO 2 .

Techniques: Injection

Journal: PLoS ONE

Article Title: Melatonin Cytotoxicity Is Associated to Warburg Effect Inhibition in Ewing Sarcoma Cells

doi: 10.1371/journal.pone.0135420

Figure Lengend Snippet: (A) Glucose uptake was measured in TC-71 cells treated with o without 1 mM melatonin for several times (2h, 4h, 6h and 8h). Data are expressed as RFU (relative fluorescence units). (B) Glucose uptake was measured in A4573 and A-673 cells treated with o without 1 mM melatonin for 8h. Data are expressed as RFU (relative fluorescence units). (C) Electron microscopy images showing the decrease in glycogen stores after treatment of Ewing sarcoma cells with 1 mM melatonin for 24 hours. Black arrows indicate glycogen stores that appear at cytoplasm forming rosettes. Also dense granules of glycogen can be observed free in the cytoplasm in control groups. N, nucleus; m, mitochondria; G, golgi apparatus areas. Right images on each experimental group correspond to a higher magnification of left image. Bars: 1μm (left image) /0.5μm (right image). (D) Intracellular glycogen levels were assessed in TC-71, A-4573 and A-673 cells after incubation with 1 mM melatonin for 4 hours. (E) Cell death was determined by means of the LDH-release assay. Ewing sarcoma cells were treated with or without 1 mM melatonin and 10 μM CP316819 for 72 hours, and cell death was calculated as the ratio between released and total LDH activity on each experimental group. Data are expressed as the percentage of control (vehicle-treated cells).*p≤0.05 vs. vehicle-treated cells; #p≤0.05 vs. melatonin-treated cells.

Article Snippet: sw-1353 (chondrosarcoma) and A-673(Ewing sarcoma) cell lines were purchased from American Type Culture Collection (Teddington, United Kingdom) and TC-71 and A-4573 (Ewing sarcoma) cell line were a generous gift from Dr J.A.

Techniques: Fluorescence, Electron Microscopy, Incubation, Lactate Dehydrogenase Assay, Activity Assay

Journal: PLoS ONE

Article Title: Melatonin Cytotoxicity Is Associated to Warburg Effect Inhibition in Ewing Sarcoma Cells

doi: 10.1371/journal.pone.0135420

Figure Lengend Snippet: (A) Lactate levels (nmoles/μl) were quantified in TC-71 cells treated with 1 mM melatonin for 2, 4, 6 and 24 hours. (B) LDH activity was evaluated in TC-71 cells incubated with 1 mM melatonin for several times (2h, 4h, 6h and 24 hours), and results were normalized with protein content (μg) in each sample. (C) Intracellular lactate levels and LDH activity were evaluated in A-4573 and A-673 Ewing sarcoma cells treated with 1 mM melatonin for 24 hours. Data are represented as percentage versus control group. (D) Cell viability (% of death cells) was evaluated by trypan blue after the incubation of TC-71, A4573 and A-673 Ewing sarcoma cells with 1mM melatonin and 16.2 mM oxamate for 48 hours. (E) Intracellular lactate levels (nmoles/μl) were quantified in sw-1353 chondrosarcoma cells treated with 1 mM melatonin for 2, 4, 6 and 24 hours. (F) LDH activity was evaluated in sw-1353 chondrosarcoma cells incubated with 1 mM melatonin for 2, 4, 6 and 24 hours, and results were normalized with protein content (μg) in each sample. *p≤0.05 vs. vehicle-treated cells; #p≤0.05 vs. melatonin-treated cells.

Article Snippet: sw-1353 (chondrosarcoma) and A-673(Ewing sarcoma) cell lines were purchased from American Type Culture Collection (Teddington, United Kingdom) and TC-71 and A-4573 (Ewing sarcoma) cell line were a generous gift from Dr J.A.

Techniques: Activity Assay, Incubation

Journal: PLoS ONE

Article Title: Melatonin Cytotoxicity Is Associated to Warburg Effect Inhibition in Ewing Sarcoma Cells

doi: 10.1371/journal.pone.0135420

Figure Lengend Snippet: Western blot analyses were carried out to identify the effect of melatonin (1 mM, 4 and 8 hours) on the activation of HIF-1α in TC-71 cells (A) and A-4573 and A-673 (1mM, 4 hours) cells (B). AKT and mTOR activation in TC-71 (C) and A-4573 and A-673 cells (D) was evaluated using specific phosphoantibodies. GAPDH was used as a loading control in all cases. A representative blot is showed. Optical density of bands was measured and values of the hydroxy-HIF-1α (inactivated form), p-AKT or p-mTOR bands were normalized versus GAPDH. Results are represented as percentage of the values found in vehicle-treated cells (dotted line). (E) Left panel , cell viability was evaluated by MTT reduction assay after treatment of TC-71 cells with 1 mM melatonin alone or in combination with 10 nM rapamycin or 10 μM LY294002 for 48 hours. Data are expressed as the percentage of vehicle-treated cells. Right panel , Representative western blot showing the relative protein level of p-AKT, p-mTOR and hydroxy-HIF-1α after 10 nM rapamycin or 10 μM LY294002 treatment during 24 hours in TC-71 cell line. p*≤0.05 vs. vehicle-treated cells.

Article Snippet: sw-1353 (chondrosarcoma) and A-673(Ewing sarcoma) cell lines were purchased from American Type Culture Collection (Teddington, United Kingdom) and TC-71 and A-4573 (Ewing sarcoma) cell line were a generous gift from Dr J.A.

Techniques: Western Blot, Activation Assay, MTT Reduction Assay