Journal: Stem Cell Research & Therapy

Article Title: Separating mouse malignant cell line (EL4) from neonate spermatogonial stem cells utilizing microfluidic device in vitro

doi: 10.1186/s13287-020-01671-1

Figure Lengend Snippet: Results of RT-PCR production of spermatogonial stem cells and EL4 cells. The expression of PLZF, GFRα-1, and Integα-6 in spermatogonial stem cells and H2Kb for EL4 cells. β-actin was included as a housekeeping gene

Article Snippet: Finally, in the present study, three main groups were designed, and by accessing the culture collection of Pasteur Institute, Tehran, Iran, the mouse acute lymphoblastic leukemia cell line EL4 was prepared.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Separating mouse malignant cell line (EL4) from neonate spermatogonial stem cells utilizing microfluidic device in vitro

doi: 10.1186/s13287-020-01671-1

Figure Lengend Snippet: Results of flow cytometry in the mixture of cancer cells and spermatogonial stem cells after microfluidic isolation. a The results obtained from cells without adding antibodies. b The results of EL4 cell composition and SSCs (in vitro tumor model). c The results of cell percentage after passing microfluidic of outer outlet. d The results of the percentage of cells associated with the inner outlet. Q1, the range of PLZF positive cells and negative H2k cells; Q2, the range of PLZF positive cells and positive H2k cells, Q3, the range of negative PLZF cells and negative H2k cells; Q4, the range for negative PLZF and positive H2k

Article Snippet: Finally, in the present study, three main groups were designed, and by accessing the culture collection of Pasteur Institute, Tehran, Iran, the mouse acute lymphoblastic leukemia cell line EL4 was prepared.

Techniques: Flow Cytometry, Isolation, In Vitro

Journal: Stem Cell Research & Therapy

Article Title: Separating mouse malignant cell line (EL4) from neonate spermatogonial stem cells utilizing microfluidic device in vitro

doi: 10.1186/s13287-020-01671-1

Figure Lengend Snippet: SSC colonies of mouse neonate spermatogonial stem cell after 2 weeks of culture in free-growth factors DMEM/F12, 1 week after primary culture, and EL4 tumor cell. a Complete colony of spermatogonium cells. b Tumor cells. Scale bars = 100 μm

Article Snippet: Finally, in the present study, three main groups were designed, and by accessing the culture collection of Pasteur Institute, Tehran, Iran, the mouse acute lymphoblastic leukemia cell line EL4 was prepared.

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: Separating mouse malignant cell line (EL4) from neonate spermatogonial stem cells utilizing microfluidic device in vitro

doi: 10.1186/s13287-020-01671-1

Figure Lengend Snippet: Part A is for the PLZF marker for SSC cells and part B is for tumor cell CD45 markers and the MERGE-shaped image of the above images and DAPI staining. Based on flow cytometry, in the first row, the control group, which contains the SSC and EL4 cell composition, is seen, and both types of cells are seen. In the 2nd row is the output from the outer inlet, which indicates a higher number of EL4 cell. The 3rd row refers to the output of the inner outlet which indicates a higher number of PLZF markers, the SSC cell

Article Snippet: Finally, in the present study, three main groups were designed, and by accessing the culture collection of Pasteur Institute, Tehran, Iran, the mouse acute lymphoblastic leukemia cell line EL4 was prepared.

Techniques: Marker, Staining, Flow Cytometry, 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 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

Journal: Oncology Letters

Article Title: Furosemide‑induced eradication of myeloblasts via the inhibition of tumor necrosis factor‑α expression in a patient with acute biphenotypic leukemia: A case report

doi: 10.3892/ol.2024.14711

Figure Lengend Snippet: Key results of the flow cytometry analysis of the peripheral blood pathological cells (red dots) leading to the diagnosis of acute biphenotypic leukemia using the EuroFlow acute leukemia orientation tube and B-cell precursor acute lymphoblastic leukemia panels. Leukemia cells were positive for both myeloid and B-lymphoid markers in (A) MPO and CD79a, (B) CD13 and TdT, and (C) CD33 and CD19. Green dots represent granulocytes, blue dots represent lymphocytes and pink dots represent monocytes. MPO, myeloperoxidase; TdT, terminal deoxynucleotidyl transferase; cy, cytoplasm; nu, nucleus; PE-A, phycoerythrin area; FITC-A, fluorescein isothiocyanate area; PE-Cy7-A, phycoerythrin-cyanine7 area.

Article Snippet: Further flow cytometric analysis using the EuroFlow B-cell precursor acute lymphoblastic leukemia (BCP-ALL) panel (Cytognos) identified the following blast characteristics: CD19 + , terminal deoxynucleotidyl transferase (TdT) + , CD10 + (weak), CD20 − , CD22 + , CD33 + , CD13 + , CD123 + and MPO + ( ).

Techniques: Flow Cytometry