human jurkat t-cell acute lymphoblastic leukemia (t-all) cells  (Interlab Inc)

Journal: PLoS Neglected Tropical Diseases

Article Title: Mitochondria and lipid raft-located F O F 1 -ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine

doi: 10.1371/journal.pntd.0005805

Figure Lengend Snippet: ( A ) L . panamensis promastigotes were untreated (Control) or treated with 10 μM edelfosine at the indicated times, and cells with disrupted ΔΨ m (DiOC 6 (3) low ) and ROS production (HE→Eth) were measured by flow cytometry. The numbers in each quadrant refer to the percentages of cells in each population. ( B ) L . panamensis promastigotes untreated (Control) and treated with edelfosine (EDLF) for 3 h were incubated with 2 μM HE and 10 μg/ml Hoechst 33342, and then analyzed by fluorescence microscopy. ( C ) L . panamensis promastigotes and ( D ) Jurkat cells were preincubated with 10 μg/ml CsA for 1 h, or with 10 mM NAC or 10 mM GSH for 2 h, and then incubated in the absence or presence of 10 μM edelfosine for 9 h. After treatment, the percentage of hypodiploid cells was analyzed by flow cytometry. Untreated control cells were run in parallel. ( E ) L . panamensis promastigotes and ( F ) Jurkat cells were preincubated with 10 μM rotenone, 5 mM malonate, 10 μM antimycin A, 1.5 mM azide, 50 μM CCCP or with 1 and 10 μM oligomycin ( L . panamensis and Jurkat cells, respectively) for 1 h, and then incubated in the absence or presence of 10 μM edelfosine for 9 h. After treatment, cells producing ROS were quantified by flow cytometry. Untreated control cells were run in parallel. Data shown are means ± SD or representative of three independent experiments performed. Asterisks denote that the differences between the indicated groups (C and D) and with control cells (E and F) are statistically significant. (*) P <0.05. (**) P <0.01.

Article Snippet: We also found that edelfosine was very efficient in promoting cell death in additional human leukemic cell lines, including human T-cell acute lymphoblastic leukemia (T-ALL) cell lines Jurkat (53.4 ± 6.2% apoptosis) and CEM-C7H2 (58.2 ± 5.9% apoptosis).

Techniques: Control, Flow Cytometry, Incubation, Fluorescence, Microscopy

Journal: PLoS Neglected Tropical Diseases

Article Title: Mitochondria and lipid raft-located F O F 1 -ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine

doi: 10.1371/journal.pntd.0005805

Figure Lengend Snippet: ( A ) L . panamensis promastigotes and T-cell leukemia Jurkat cells were untreated (Control) or pretreated with MCD, and then incubated in the absence or presence of 10 μM edelfosine for 24 h. Percentage of hypodiploid cells were measured by flow cytometry. ( B ) L . panamensis promastigotes and T-cell leukemia Jurkat cells were untreated (Control) or pretreated with MCD and then incubated with 10 μM [ 3 H]edelfosine for 1 h. Drug uptake was determined as shown in the Materials and Methods section. Data shown are means ± SD of three independent experiments performed. Asterisks denote that the differences between the indicated groups are statistically significant. (**) P <0.01. (***) P <0.001.

Article Snippet: We also found that edelfosine was very efficient in promoting cell death in additional human leukemic cell lines, including human T-cell acute lymphoblastic leukemia (T-ALL) cell lines Jurkat (53.4 ± 6.2% apoptosis) and CEM-C7H2 (58.2 ± 5.9% apoptosis).

Techniques: Control, Incubation, Flow Cytometry

Journal: PLoS Neglected Tropical Diseases

Article Title: Mitochondria and lipid raft-located F O F 1 -ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of ether lipid edelfosine

doi: 10.1371/journal.pntd.0005805

Figure Lengend Snippet: ( A ) Jurkat cells untreated (Control) and treated with 10 μM edelfosine for 9 h were lysed in 1% Triton X-100 and subjected to discontinuous sucrose density gradient centrifugation. Individual fractions were subjected to SDS-PAGE, and location of GM1 was determined using CTx B subunit conjugated with horseradish peroxidase. ( B ) Proteins from lipid rafts of untreated control and edelfosine-treated Jurkat cells were subjected to two-dimensional gel electrophoresis followed by MALDI-TOF analysis. Mitochondrial F O F 1 -ATP synthase β subunit is indicated by an arrow. ( C ) Mass spectrum of the tryptic peptides of the F O F 1 -ATP synthase β subunit spot. Mass value (m/z) and putative amino acid position assignments are indicated above peaks. ( Inset ) Peptide coverage map of human F O F 1 -ATP synthase β subunit; the peptides used for identification are highlighted in bold characters and underlined. ( D ) Jurkat cells were untreated (Control) or preincubated with 10 μM oligomycin for 1 h and then incubated in the absence or presence of 10 μM edelfosine for 9 h, and ΔΨ m disruption (Low ΔΨ m ) and DNA breakdown (hypodiploids) were evaluated. Data shown are means ± SD or representative of three independent experiments. Asterisks denote that the differences between the indicated groups are statistically significant. (**) P <0.01.

Article Snippet: We also found that edelfosine was very efficient in promoting cell death in additional human leukemic cell lines, including human T-cell acute lymphoblastic leukemia (T-ALL) cell lines Jurkat (53.4 ± 6.2% apoptosis) and CEM-C7H2 (58.2 ± 5.9% apoptosis).

Techniques: Control, Gradient Centrifugation, SDS Page, Two-Dimensional Gel Electrophoresis, Electrophoresis, Incubation, Disruption

Journal: BMC Cancer

Article Title: EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition

doi: 10.1186/s12885-015-1845-1

Figure Lengend Snippet: FK866 affects NAD + (H) and ATP levels in Jurkat cells leading to cell death. a Flow-cytometric quantification of cell viability with AnnexinV (FITC) and 7AAD (PerCP-Cy5-5-A) staining. Jurkat cells were treated with FK866 for 48,72 and 120 h. Mock, 5 nM FK866 and 100 nM FK866 (at 48 and 72 h) are shown as representative samples. Flow-cytometry experiments were carried out on two biological replicates and statistics were based on the acquisition of 10000 events/sample. b Cell-cycle analysis with PI staining of the nuclei after 48 h of treatment. Overnight serum starvation is shown as positive control of induced cell cycle synchronization in G0/G1 phase. Histograms quantify the cell cycle phase distribution. Flow-cytometry experiments were carried out on two biological replicates and statistics were based on acquisition of 30000 events/sample. Cell cycle phase analysis was done using ModFit LT 3.2 software and the Sync Wizard model. c Jurkat cells were treated with FK866 for 48 h. Alternatively, cells were exposed to 5 μM of Camptothecin for 4 h as a positive control. Protease activity in cell extracts was assessed with a commercially available kit and values were normalized to the protein concentration in the same extracts. d Caspase 3/7 activity was measured in Jurkat cells treated as in c . e Jurkat cells were treated with FK866 for 48 h. Thereafter, intracellular NAD + (H) and ATP levels were evaluated in comparison with control Jurkat cells. RLUs were normalized to number of viable cells. In c - e the means with SD of at least three independent experiments are shown. Statistical significance was calculated with t -test (* and # indicates p -value <0.05)

Article Snippet: Human Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells were purchased from the InterLab Cell Line Collection bank (ICLC HTL01002).

Techniques: Staining, Flow Cytometry, Cell Cycle Assay, Positive Control, Software, Activity Assay, Protein Concentration, Comparison, Control

Journal: BMC Cancer

Article Title: EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition

doi: 10.1186/s12885-015-1845-1

Figure Lengend Snippet: FK866 inhibits the signaling cascades controlling protein synthesis in T-ALL cell lines. a RNA synthesis was determined by monitoring EU incorporation with Click-it chemistry. Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of FK866 or for 3 h with 5 μM Actinomycin D, an RNA synthesis blocking agent. The histogram quantifies the dose-dependent transcription inhibition induced by FK866 in the viable cell population. In the lower part, Click-it chemistry based on the incorporation of an aminoacid analog (AHA) was used to monitor protein synthesis. Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of FK866 or for 3 h with 350 μM Cycloheximide, as a positive control for protein synthesis inhibition. The histogram quantifies FK866-induced protein synthesis arrest in the viable cell population. Flow-cytometry experiments were carried out on two biological replicates and statistics were based on acquisition of 50000 events/sample. b Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of FK866. Thereafter, cells were lysed and the levels of total and p-Akt (Ser-473), total and p-MTOR (Ser-2448), total and p-4EBP1 (Ser-65 and Thr-70), c ) total and p-EIF4E (Ser-209), total and p-EIF2A (Ser-51) were detected by immunoblotting. d Molt-4 cells were treated with FK866 for 48 h and the levels of total 4EBP1 and p-4EBP1 were evaluated. e Western blot analysis as in d in SupT1 cells. b - e , one representative experiment out of at least three biological replicates is presented and β-actin was used as loading control

Article Snippet: Human Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells were purchased from the InterLab Cell Line Collection bank (ICLC HTL01002).

Techniques: Concentration Assay, Blocking Assay, Inhibition, Positive Control, Flow Cytometry, Western Blot, Control

Journal: BMC Cancer

Article Title: EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition

doi: 10.1186/s12885-015-1845-1

Figure Lengend Snippet: FK866 induces AMPK and EIF2A phosphorylation in primary leukemia cells. a Jurkat cells were treated for 48 h with or without (Mock) the indicated concentration of FK866. Thereafter, cells were lysed and the levels of total and p-AMPK (Thr-172), ACC and p-ACC, BCL-2, MCL1 and β-actin as loading control were detected by immunoblotting. One representative experiment out of three biological replicates. b primary B-CLL cells (source: peripheral blood; RAI stage III, 86 years, CD38-pos) were treated for 48 h with or without FK866 in the presence or absence of 1 mM NA. Thereafter, protein lysates were immunoblotted for AMPK and p-AMPK. c Cell viability with respect to Mock condition, measured by CellTiter Glo, of three different T-ALL xenografts (PD T-ALL 12, 19 and 25) after treatment with FK866 5nM for 48 h. d WB of PD T-ALL 12 as representative of T-ALL xenografts samples. Cells were treated with 5 and 50 nM FK866 for 48 h. Histogram shows the densitometric analysis of p-AMPK and p-EIF2A in the three T-ALL xenografts (PD T-ALL 12, 19 and 25)

Article Snippet: Human Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells were purchased from the InterLab Cell Line Collection bank (ICLC HTL01002).

Techniques: Phospho-proteomics, Concentration Assay, Control, Western Blot

Journal: BMC Cancer

Article Title: EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition

doi: 10.1186/s12885-015-1845-1

Figure Lengend Snippet: NAMPT genetic ablation induces EIF2A phosphorylation and MCL1 down-regulation. a Expression levels of NAMPT. Jurkat cells were treated with 5 and 100 nM FK866 for 48 h. One representative experiment out of three biological replicates is presented. b WB analysis indicated 50 % of NAMPT silencing ( p < 0.001) in Jurkat cells by using lentiviral particles expressing two NAMPT-silencing shRNAs (shNAMPT-1 and −2). c Intracellular NAD + (H) and ATP levels in shNAMPT cells (transduced with shNAMPT-1 and −2) were evaluated in comparison with scramble Jurkat cells. Thirty percent reduction of NAD + (H) levels was observed in shNAMPT cells ( p -value < 0.01). RLUs were normalized to number of viable cells. Mean and SD of a biological triplicate. d WB analysis of AMPK, p-AMPK, EIF2A, p-EIF2A, p-4EBP1 and MCL1 in shNAMPT (transduced with shNAMPT-1 and −2) cells. Histogram shows the densitometric analysis of p-AMPK, p-EIF2A and MCL1 (* indicates p -value <0.05). Mean and SD of a biological triplicate

Article Snippet: Human Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells were purchased from the InterLab Cell Line Collection bank (ICLC HTL01002).

Techniques: Phospho-proteomics, Expressing, Transduction, Comparison

Journal: BMC Cancer

Article Title: EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition

doi: 10.1186/s12885-015-1845-1

Figure Lengend Snippet: AMPK regulates EIF2A phosphorylation and is a pro-survival factor in Jurkat cells. a Jurkat cells were treated with or without FK866 at the indicated concentrations in the presence or absence of Compound C 5 μM for 48 h. Thereafter, cells were lysed and the levels of p-AMPK (Thr-172), p-MTOR, 4EBP1, p-4EBP1, BCL-2, MCL1, EIF2A and p-EIF2A were revealed by immunoblotting. Histogram shows the densitometric analysis of p-AMPK and p-EIF2A. b In the same samples, caspase 3/7 activity was quantified and relative ATP levels were determined and then normalized to the number of viable cells (* indicates p -value <0.05). a and b are representative of a biological triplicate (mean and SD). c Jurkat cells were transduced with lentiviral particles containing scramble or shAMPK (targeting the α1 and the α2 subunit), then treated for 48 h with or without 5 nM FK866. Cell lysates were used for total AMPK, EIF2A, p-EIF2A, 4EBP1, p-4EBP1 and β-actin immunoblotting. WB analysis indicated 40 % of AMPK silencing ( p -value < 0.05) and densitometric analysis shows the significant decrease of p-EIF2A in shAMPK Jurkat cells ( p -value < 0.03). Mean and SD of a biological triplicate. d Jurkat scramble and shAMPK cells were treated for 48 h with the indicated doses of FK866. Cell viability was determined by PI staining and flow-cytometry (two biological replicates and statistics based on the acquisition of 10000 events/sample)

Article Snippet: Human Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells were purchased from the InterLab Cell Line Collection bank (ICLC HTL01002).

Techniques: Phospho-proteomics, Western Blot, Activity Assay, Transduction, Staining, Flow Cytometry

Journal: BMC Cancer

Article Title: EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition

doi: 10.1186/s12885-015-1845-1

Figure Lengend Snippet: Protective role of EIF2A and FK866 induced UPR. a Expression level of LKB1 mRNA, evaluated in Jurkat cells after 120 h of lentiviral transduction with shRNAs expressing the control sequence (scramble) or two LKB1-silencing shRNAs (shLKB1- a and – b ), in the upper panel. Cells were transduced for 72 h and then treated with FK866 for 48 h. Viability was measured by MTT assay in comparison with Mock (DMSO) condition, in the lower panel. Mean and SD of a biological triplicate (*, p -value < 0.05). b WB analysis indicated the levels of AMPK, p-AMPK, p-EIF2A, p-4EBP1 in A594 cells expressing LKB1 (LKB1 WT) or transduced with an empty vector (pBABE) treated or not (Mock) with 100 nM FK866 for 48 h, left panel. A549 cells were treated with indicated concentration of FK866 for 48 h and cell viability as shown in dose–response curve was evaluated by MTT assay, right panel. Mean and SD of three biological replicates. c A549 viability after 48 h of treatment with FK866 100 nM in un-transfected (NTC) cells and transfected with EIF2A wild type, EIF2A-S51A, EIF2A-S51D (mean and SD of three experiments,*, p -value < 0.05). d Expression level of BiP mRNA, evaluated in Jurkat cells after 48 h of treatment with FK866 5nM (*, p -value < 0.0005). Mean and SD of a biological triplicate. e WB analysis of MCL1 in Jurkat cells treated with FK866 for 48 h and with the proteasome inhibitor MG132 1 μM for 24 h. MG132 was added after 24 h of FK866 treatment. One representative experiment out of three biological replicates

Article Snippet: Human Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells were purchased from the InterLab Cell Line Collection bank (ICLC HTL01002).

Techniques: Expressing, Transduction, Control, Sequencing, MTT Assay, Comparison, Plasmid Preparation, Concentration Assay, Transfection