Journal: Technology in Cancer Research & Treatment

Article Title: Thymoquinone and Difluoromethylornithine (DFMO) Synergistically Induce Apoptosis of Human Acute T Lymphoblastic Leukemia Jurkat Cells Through the Modulation of Epigenetic Pathways

doi: 10.1177/1533033820947489

Figure Lengend Snippet: Effect of Thymoquinone and/or DFMO on Jurkat cell viability. Cells were exposed to increasing concentrations of DFMO (A) or TQ (B) for 24 h. To evaluate the synergistic effect on cell viability, cells were treated with either DFMO (1 mM) for 48 h or TQ (10 μM) or incubated with 1 mM of DFMO for 24 h before adding 10 μM of TQ for additional 24 h (C). Cell viability rate was assessed by WST-1 assay, as indicated in the methods and materials. The data are representative of 3 different experiments. Values are shown as means ± S.E.M. (n = 3); *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001, ## , p < 0.01, && , p < 0.01 versus respective control.

Article Snippet: Jurkat cell viability rate was also determined by cell counting using the trypan blue exclusion method (Invitrogen).

Techniques: Incubation, WST-1 Assay, Control

Journal: Technology in Cancer Research & Treatment

Article Title: Thymoquinone and Difluoromethylornithine (DFMO) Synergistically Induce Apoptosis of Human Acute T Lymphoblastic Leukemia Jurkat Cells Through the Modulation of Epigenetic Pathways

doi: 10.1177/1533033820947489

Figure Lengend Snippet: DFMO and Thymoquinone synergize to induce apoptosis in Jurkat cells. To evaluate the synergistic effect on apoptosis, cells were treated with either DFMO at 1 mM for 48 h or TQ at 10 μM or incubated with 1 mM of DFMO for 24 h before adding TQ at (10 μM) for additional 24 h (A & B). To confirm the synergistic effect of TQ and DFMO, cells were treated with either DFMO at 0.5 mM for 48 h or TQ at 20 μM or incubated with 0.5 mM of DFMO for 24 h before adding TQ at (20 μM) for additional 24 h. Apoptosis in Jurkat cells was assessed by flow cytometry using the Annexin V/7AAD staining apoptosis assay (A, B, C & D). Values are shown as means ± S.E.M. (n = 3); *, p < 0.05, ***, p < 0.001, ****, p < 0.0001, ### , p < 0.001, #### , p < 0.0001, && , p < 0.01, &&&& , p < 0.0001 versus respective control.

Article Snippet: Jurkat cell viability rate was also determined by cell counting using the trypan blue exclusion method (Invitrogen).

Techniques: Incubation, Flow Cytometry, Staining, Apoptosis Assay, Control

Journal: Technology in Cancer Research & Treatment

Article Title: Thymoquinone and Difluoromethylornithine (DFMO) Synergistically Induce Apoptosis of Human Acute T Lymphoblastic Leukemia Jurkat Cells Through the Modulation of Epigenetic Pathways

doi: 10.1177/1533033820947489

Figure Lengend Snippet: Downregulated Genes Triggered in DFMO-Treated Jurkat Cells as Compared with Untreated Cells.

Article Snippet: Jurkat cell viability rate was also determined by cell counting using the trypan blue exclusion method (Invitrogen).

Techniques: Gene Expression, Ubiquitin Proteomics, Histone Deacetylase Assay

Journal: Technology in Cancer Research & Treatment

Article Title: Thymoquinone and Difluoromethylornithine (DFMO) Synergistically Induce Apoptosis of Human Acute T Lymphoblastic Leukemia Jurkat Cells Through the Modulation of Epigenetic Pathways

doi: 10.1177/1533033820947489

Figure Lengend Snippet: Upregulated Tumor Suppressor Genes in DFMO-Treated Jurkat Cells as Compared With Untreated Cells.

Article Snippet: Jurkat cell viability rate was also determined by cell counting using the trypan blue exclusion method (Invitrogen).

Techniques:

Journal: Technology in Cancer Research & Treatment

Article Title: Thymoquinone and Difluoromethylornithine (DFMO) Synergistically Induce Apoptosis of Human Acute T Lymphoblastic Leukemia Jurkat Cells Through the Modulation of Epigenetic Pathways

doi: 10.1177/1533033820947489

Figure Lengend Snippet: Upregulated Pro-Apoptotic Genes in DFMO-Treated Jurkat Cells as Compared With Untreated Cells.

Article Snippet: Jurkat cell viability rate was also determined by cell counting using the trypan blue exclusion method (Invitrogen).

Techniques:

Journal: Technology in Cancer Research & Treatment

Article Title: Thymoquinone and Difluoromethylornithine (DFMO) Synergistically Induce Apoptosis of Human Acute T Lymphoblastic Leukemia Jurkat Cells Through the Modulation of Epigenetic Pathways

doi: 10.1177/1533033820947489

Figure Lengend Snippet: Heat map of the deregulated genes in treated versus control cells. The signature of the deregulated genes are represented in the intensity of color; with the alteration of LogFC (fold change) from -1 to +3 in DFMO-treated Jurkat cells as compared to the untreated cells.

Article Snippet: Jurkat cell viability rate was also determined by cell counting using the trypan blue exclusion method (Invitrogen).

Techniques: Control

Journal: Technology in Cancer Research & Treatment

Article Title: Thymoquinone and Difluoromethylornithine (DFMO) Synergistically Induce Apoptosis of Human Acute T Lymphoblastic Leukemia Jurkat Cells Through the Modulation of Epigenetic Pathways

doi: 10.1177/1533033820947489

Figure Lengend Snippet: Synergistic effect of TQ and DFMO on the expression of UHRF1, DNMT1 and HDAC1 mRNA levels in Jurkat cells. To evaluate the synergistic effect on the expression of UHRF1, DNMT1 and HDAC1 genes, cells were treated with either DFMO (1 mM) for 48 h or TQ (10 μM) or incubated with 1 mM of DFMO for 24 h before adding 10 μM of TQ for additional 24 h. The histograms show the quantification data of mRNA expressions of UHRF1 (A), DNMT1 (B) and HDAC1 (C), as assessed by real-time PCR. Results are means of 3 separate experiments performed in triplicate. Values are shown as means ± S.E.M. (n = 3); *, p < 0.05, ***, p < 0.001, ****, p < 0.0001, ## , p < 0.01, ### , p < 0.001, &&& , p < 0.001, ### , p < 0.001, &&&& , p < 0.0001 versus respective control.

Article Snippet: Jurkat cell viability rate was also determined by cell counting using the trypan blue exclusion method (Invitrogen).

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Control

Journal: Technology in Cancer Research & Treatment

Article Title: Thymoquinone and Difluoromethylornithine (DFMO) Synergistically Induce Apoptosis of Human Acute T Lymphoblastic Leukemia Jurkat Cells Through the Modulation of Epigenetic Pathways

doi: 10.1177/1533033820947489

Figure Lengend Snippet: Effect of the depletion of UHRF1 cell viability. Jurkat cells were transfected with siRNA against UHRF1 for 72 h. (A): Western blot was then performed using an anti-UHRF1 antibody as described in materials and methods. (B): Cell viability was calculated using trypan blue as indicated in materials and methods. Data are shown as mean ± SE of 3 independent experiments ( #### P < 0.0001, ***P < 0.001 versus respective control).

Article Snippet: Jurkat cell viability rate was also determined by cell counting using the trypan blue exclusion method (Invitrogen).

Techniques: Transfection, Western Blot, Control

Journal: iScience

Article Title: Necrosulfonamide causes oxidation of PCM1 and impairs ciliogenesis and autophagy

doi: 10.1016/j.isci.2024.109580

Figure Lengend Snippet: Necrosulfonamide causes the oxidation and aggregation of PCM1 independently of MLKL (A) Densitometric measurement of PCM1 abundance, normalized to GAPDH and compared to DMSO-treated samples analyzed by western blotting in Jurkat cells treated overnight with 35 small-molecule compounds. The inset blot shows samples from cells treated with NSA (2.5 μM), or with DMSO overnight analyzed by western blotting as indicated. Molecular weight markers ( Mr ) are indicated. PCM1 n indicates high-molecular-weight species of PCM1. (B) CellTiter-Glo Luminescent cell viability assay of Jurkat cells treated overnight with 2.5 or 5 μM NSA (biological triplicates, ∗∗∗ p < 0.001, ANOVA). (C) RNA-seq transcriptomic analysis of Jurkat cells treated with DMSO or 2.5 μM NSA. The scatter (volcano) plot shows Log2 Fold Change and -Log10 (padj); n = 3. Dashed lines, significance cut-off. Purple symbols indicate differentially expressed genes associated with the main function “oxidoreductase activity”. (D) Flow cytometry analysis of ROS using CellROX in Jurkat cells pretreated with 5 mM N-acetylcysteine (NAC) for 2 h and exposed to 2.5 μM NSA overnight (mean ± SEM; n = 3; ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ANOVA). (E) Jurkat cells were pretreated with 5 mM NAC for 2 h before overnight incubation with NSA, as indicated. Cell lysates were analyzed by Western blotting in reducing (10% 2β-ME) and non-reducing conditions. Aggre., aggregates; mono., monomeric. (F) Jurkat cells were treated with 2.5 μM NSA or DMSO, as indicated. Cell lysates were analyzed by Western blotting in non-reducing conditions. (G) Jurkat cells were pretreated with 10 nM bortezomib (BTZ) or 100 nM bafilomycin A1 (BafA1) for 1 h and incubated with 2.5 μM NSA overnight. Cell lysates were analyzed by western blotting with antibodies specific to the indicated proteins. (H) L929 cells were treated and analyzed as in (E). All presented data are representative of three independent experiments.

Article Snippet: Human: Jurkat cell line (clone E6.1) , ATCC , TIB-152.

Techniques: Western Blot, Molecular Weight, High Molecular Weight, Cell Viability Assay, RNA Sequencing, Activity Assay, Flow Cytometry, Incubation

Journal: iScience

Article Title: Necrosulfonamide causes oxidation of PCM1 and impairs ciliogenesis and autophagy

doi: 10.1016/j.isci.2024.109580

Figure Lengend Snippet: Necrosulfonamide inhibits ciliogenesis (A–C) Confocal microscopy analysis of Jurkat cells treated overnight with 2.5 μM NSA showing the localization of PCM1 and γ-tubulin, with nuclei stained by 4′-6-diamidino-2-phenylindole (DAPI). Scale bar, 2 μm. The radial profile (B) and the mean intensity (C) of PCM1 around the centrosome defined by γ-tubulin were measured ( n = 37 (DMSO) and n = 48 (NSA) cells from of three independent experiments; ns, non-significant; ANOVA). (D) Jurkat cells were pretreated with 5 mM N-acetylcysteine (NAC) for 2 h and incubated with 2.5 μM NSA overnight. Cell lysates prepared in non-reduced conditions were analyzed by western blotting with antibodies specific to the indicated proteins. Molecular weight markers ( Mr ) are indicated. Aggre., aggregates; mono., monomeric. (E) Cell lysates from Jurkat cells treated with 2.5 μM NSA overnight were subjected to immunoprecipitation (IP) with antibodies against PCM1 or with control (ctrl) antibodies, and samples were then analyzed by wstern blotting as indicated. (F and G) RPE-1 cells were serum starved and treated with 2.5 μM NSA with and without NAC (5 mM) for 24 h and were then analyzed by confocal microscopy to visualize PCM1 and primary cilia (acetylated tubulin, arrowheads). Scale bar, 20 μm. In (F), the histogram shows the quantification of cells with cilia (mean ± SEM of three independent experiments; n > 100 cells counted per sample; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ANOVA). All presented data are representative of three independent experiments.

Article Snippet: Human: Jurkat cell line (clone E6.1) , ATCC , TIB-152.

Techniques: Confocal Microscopy, Staining, Incubation, Western Blot, Molecular Weight, Immunoprecipitation, Control

Journal: iScience

Article Title: Necrosulfonamide causes oxidation of PCM1 and impairs ciliogenesis and autophagy

doi: 10.1016/j.isci.2024.109580

Figure Lengend Snippet: Necrosulfonamide causes a defect in autophagy (A) Cellular Component enrichment analysis of differentially expressed genes analyzed from an RNA-seq analysis of Jurkat cells treated overnight with 2.5 μM NSA, as described in Figure 1 C. (B) Volcano plot of RT 2 profiler PCR array of human autophagy signaling components for Jurkat cells treated as in (A). Data represent three independent experiments. Genes upregulated upon NSA treatment are shown. (C) Jurkat cells pre-incubated with 5 mM N-acetylcysteine (NAC) were treated with 2.5 μM NSA overnight. Cell lysates prepared in non-reduced conditions were analyzed by western blotting with antibodies specific to the indicated proteins. Aggre., aggregated proteins; mono., monomeric proteins. Molecular weight markers ( Mr ) are indicated. (D and E) Confocal microscopy analysis of p62 in RPE-1 cells pretreated with 100 nM bafilomycin A1 (BafA1) for 1 h and incubated with 2.5 μM NSA overnight. Nuclei were stained by DAPI. Scale bar, 10 μm. The number of p62 puncta per cell is shown in (E) ( n > 96 cells analyzed per sample of four independent experiments, ∗∗∗∗ p < 0.0001, t test). (F and G) Confocal microscopy analysis of GABARAPL1 in RPE-1 cells as in (D and E) ( n > 113 cells analyzed per sample of four independent experiments, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, t test). (H) Cell lysates from Jurkat cells treated with 2.5 μM of NSA as indicated were prepared in non-reduced conditions and analyzed by western blotting with antibodies against the indicated proteins. (I) Jurkat cells were transfected with small interfering RNA (siRNA) for PCM1 or scramble non-specific (NS). Cells were treated overnight with NSA as indicated. Cell lysates were prepared and analyzed by western blotting with antibodies specific against the indicated proteins. All presented data are representative of three independent experiments.

Article Snippet: Human: Jurkat cell line (clone E6.1) , ATCC , TIB-152.

Techniques: RNA Sequencing, Incubation, Western Blot, Molecular Weight, Confocal Microscopy, Staining, Transfection, Small Interfering RNA

Journal: iScience

Article Title: Necrosulfonamide causes oxidation of PCM1 and impairs ciliogenesis and autophagy

doi: 10.1016/j.isci.2024.109580

Figure Lengend Snippet:

Article Snippet: Human: Jurkat cell line (clone E6.1) , ATCC , TIB-152.

Techniques: Recombinant, Transfection, Electron Microscopy, Protease Inhibitor, Western Blot, Purification, cDNA Synthesis, SYBR Green Assay, Viability Assay, Sequencing, Software, Imaging

Journal: Journal of Nanobiotechnology

Article Title: Comprehensive and systematic characterization of multi-functionalized cisplatin nano-conjugate: from the chemistry and proteomic biocompatibility to the animal model

doi: 10.1186/s12951-022-01546-y

Figure Lengend Snippet: A Viability assay performed in Caco-2 and Jurkat cancer cell lines using Pt precursor, IONP-Pt and IONP-Pt-Flu at different concentrations (1 and 0.1 mg/mL) incubating for 24–72 h. Results are reported as the mean number of live cells relative to the control (vehicle) from three independent experiments (significantly differences p * < 0.05, ** < 0.01). B Flow cytometry results (apoptosis and cell cycle assays) of NPs tested (IONP-Pt and IONP-Pt-Flu) for 24 h in Caco-2 cell line and Jurkat cancer cell lines at 0.1 mg/mL. Percentage of apoptotic cells (annexin+) and G2/M cells of each experiment are shown in the corresponding diagram as the mean of two independent experiments for each condition

Article Snippet: All the cell lines [Jurkat, T-cell leukemia (DSMZ ACC 282); Caco-2 (ATCC® HTB-37TM)] were cultured at 37 °C in a humidified CO2 incubator (5% CO 2 ) in complete RPMI media (Jurkat) or DMEM media (Caco-2) [(RPMI-1640 medium or DMEM supplemented with 10% (v/v) FBS and 1% (v/v) P-S].

Techniques: Viability Assay, Control, Flow Cytometry

Journal: Journal of Nanobiotechnology

Article Title: Comprehensive and systematic characterization of multi-functionalized cisplatin nano-conjugate: from the chemistry and proteomic biocompatibility to the animal model

doi: 10.1186/s12951-022-01546-y

Figure Lengend Snippet: A Functional characterization pipeline illustration and summary. Left Venn diagram depicts the common proteins synthesized at different NP conditions and right venn diagram the respective functional enrichment similarities after functional semantic simplification. B NP-induced newly synthesized proteome overview and overlap analysis. Bar plots and venn diagrams showing the number of proteins commonly synthesized at control conditions and NP-Cis-Pt or NP-Cis-PT-FITC presence at the experiments conducted on Caco-2 ( A ) and Jurkat ( B ) tumor cell lines respectively

Article Snippet: All the cell lines [Jurkat, T-cell leukemia (DSMZ ACC 282); Caco-2 (ATCC® HTB-37TM)] were cultured at 37 °C in a humidified CO2 incubator (5% CO 2 ) in complete RPMI media (Jurkat) or DMEM media (Caco-2) [(RPMI-1640 medium or DMEM supplemented with 10% (v/v) FBS and 1% (v/v) P-S].

Techniques: Functional Assay, Synthesized, Control

Journal: Journal of Nanobiotechnology

Article Title: Comprehensive and systematic characterization of multi-functionalized cisplatin nano-conjugate: from the chemistry and proteomic biocompatibility to the animal model

doi: 10.1186/s12951-022-01546-y

Figure Lengend Snippet: Analysis summarizing functional simplification in Caco-2 and Jurkat cell line. Each function color indicates the resulting functional group according to REVIGO method

Article Snippet: All the cell lines [Jurkat, T-cell leukemia (DSMZ ACC 282); Caco-2 (ATCC® HTB-37TM)] were cultured at 37 °C in a humidified CO2 incubator (5% CO 2 ) in complete RPMI media (Jurkat) or DMEM media (Caco-2) [(RPMI-1640 medium or DMEM supplemented with 10% (v/v) FBS and 1% (v/v) P-S].

Techniques: Functional Assay

Journal: Molecular cancer therapeutics

Article Title: Ref-1/APE1 as Transcriptional Regulator and Novel Therapeutic Target in Pediatric T-cell Leukemia

doi: 10.1158/1535-7163.MCT-17-0099

Figure Lengend Snippet: A) Cell death as defined by the Annexin V/PI assay in leukemia TAIL7 cells treated with E3330 at the doses indicated. Data is shown as mean ± SD. *p<0.05, **p<0.01, using T-test. B) qPCR for mRNA expression of Survivin/BIRC5, Bcl-xL in TAIL7 cells treated with E3330, at the doses indicated. GAPDH was used as endogenous control, and the assays performed using TaqMan probes. Data shown as mean ± SEM, n=4; *p<0.05, **p<0.01, using 1-way ANOVA. C) ATP viability assay with E3330 in Jurkat cells overexpressing Bcl-2 (Jurkat/Bcl2) in comparison to control, vector-expressing cells (Jurkat/Neo); analyses at 96hrs. Data shown as mean ± SEM, from 4 independent experiments; *p<0.05, **p<0.01, for Jurkat/Bcl2 vs. Jurkat/Neo, using 2-way ANOVA.

Article Snippet: The Jurkat/Bcl2 and Jurkat/Neo are sublines derived from transfection of Jurkat cells respectively with a psFFV-neo expressing vector containing human BCL2 or empty vector, and were obtained from ATCC in 2014.

Techniques: Expressing, Control, Viability Assay, Comparison, Plasmid Preparation

Journal: Scientific reports

Article Title: 6E11, a highly selective inhibitor of Receptor-Interacting Protein Kinase 1, protects cells against cold hypoxia-reoxygenation injury.

doi: 10.1038/s41598-017-12788-4

Figure Lengend Snippet: Figure 2. Characterization of hit compound 6E11 as new necroptosis inhibitor. (a) Workflow of the cell- based screening of ICBMS chemical library for the selection of new inhibitors of necroptosis. Among 2,800 compounds, 6E11, was selected as the more potent inhibitor of TNF-α-induced necroptosis in human FADD- deficient Jurkat T cells. The chemical structures of 6E11 and its negative control (8A03) are depicted above the workflow. The primary screening is performed in monoplicate. The negative control was not detected during the screening campaign. (b) Dose-dependent protection of 6E11 against TNF-α-induced Jurkat FADD deficient cell necroptosis. After a 24-h incubation of the cells with or without (w/o) TNF-α and increasing concentrations of tested compounds, the effect on the cell viability was evaluated by MTS reduction assay. The cells were treated only with the tested compound to evaluate its putative toxicity. The values were normalized as a percentage of cell viability, considering 100% viable cells in the control treated with DMSO (n = 3, mean ± SD).

Article Snippet: Jurkat wild-type A3, FADD-deficient Jurkat I 2.1 and RPE-1 hTERT human cell lines were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA).

Techniques: Selection, Negative Control, Incubation, Control

Journal: Scientific reports

Article Title: 6E11, a highly selective inhibitor of Receptor-Interacting Protein Kinase 1, protects cells against cold hypoxia-reoxygenation injury.

doi: 10.1038/s41598-017-12788-4

Figure Lengend Snippet: Figure 3. 6E11 inhibits death receptor-induced necroptosis, but not apoptosis. (a) Human FADD-deficient Jurkat T cells were treated or not with TNF-α (10 ng/ml) in presence or not of increasing concentrations of 6E11 (0, 1, 5, 10 µM) or Nec-1 (0, 5, 10 µM) for 18 hours. Intracellular ATP levels were measured with the CellTiter-Glo® Luminescent Cell Viability Assay (n = 3, mean ± SEM, *P < 0.05) (EC50 ~6 µM). (b) Human FADD-deficient Jurkat T cells were treated or not with TNF-α (10 ng/ml) in presence or not of increasing concentrations of 6E11 (0, 5, 10, 20 µM) or Nec-1 (0, 5, 10 µM) for 18 hours. The percentage of cell death was determined by propidium iodide staining using flow cytometry (n = 3, mean ± SEM, **P < 0.01 and ***P < 0.001). (c) Human FADD-deficient Jurkat T cells were treated or not with TNF-α (10 ng/ml) in presence or not of increasing concentrations of 6E11 (0, 5, 10, 20 µM) for 18 hours. The mitochondrial transmembrane potential (MTP) was measured using the fluorescent dye DiOC6(3) and flow cytometry analysis. (d) 6E11 inhibits TRAIL-induced necroptosis. Wild-type Jurkat T cells were treated or not with TRAIL (10 ng/ml), Z-VAD (30 µM) and CHX (1 µg/ml) for 18 hours in presence or not of increasing concentrations of 6E11 (0, 5, 10, 20 µM) or 10 µM Nec-1. The percentage of cell death was determined by propidium iodide staining using flow cytometry (n = 3, mean ± SEM, **P < 0.01). (e) 6E11 does not inhibit TRAIL-induced apoptosis. Wild- type Jurkat T cells were treated or not with TRAIL (20 ng/ml) for 18 hours in presence or not of increasing concentrations of 6E11 (0, 5, 10, 20 µM) or Nec-1 (0, 5, 10, 20 µM). The percentage of cell death was determined by propidium iodide staining using flow cytometry (n = 3, mean ± SEM).

Article Snippet: Jurkat wild-type A3, FADD-deficient Jurkat I 2.1 and RPE-1 hTERT human cell lines were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA).

Techniques: Cell Viability Assay, Staining, Flow Cytometry

Journal: Scientific reports

Article Title: 6E11, a highly selective inhibitor of Receptor-Interacting Protein Kinase 1, protects cells against cold hypoxia-reoxygenation injury.

doi: 10.1038/s41598-017-12788-4

Figure Lengend Snippet: Figure 4. 6E11 is not cytotoxic at inhibitory concentration and protects from necroptosis even after cell death initiation. (a) 24 hours after treatment with increasing concentrations of 6E11, cell viability was measured with a MTS assay to determine the toxicity of the compound towards either human PBL (upper panel) (n = 6 individuals, mean ± SEM) or human retina pigmented epithelial cells (RPE-1 hTERT) (lower panel) (n = 3, mean ± SD). (b) Viability was measured with a MTS assay in FADD-def Jurkat cells treated with TNF-α (10 ng/ ml) followed by 10 µM 6E11 addition 1 to 4 h post necroptosis initiation (n = 4, mean ± SEM, *P < 0.05; **P < 0.01).

Article Snippet: Jurkat wild-type A3, FADD-deficient Jurkat I 2.1 and RPE-1 hTERT human cell lines were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA).

Techniques: Concentration Assay, MTS Assay

Journal: Molecular cancer therapeutics

Article Title: Ref-1/APE1 as Transcriptional Regulator and Novel Therapeutic Target in Pediatric T-cell Leukemia

doi: 10.1158/1535-7163.MCT-17-0099

Figure Lengend Snippet: A) Cell death as defined by the Annexin V/PI assay in leukemia TAIL7 cells treated with E3330 at the doses indicated. Data is shown as mean ± SD. *p<0.05, **p<0.01, using T-test. B) qPCR for mRNA expression of Survivin/BIRC5, Bcl-xL in TAIL7 cells treated with E3330, at the doses indicated. GAPDH was used as endogenous control, and the assays performed using TaqMan probes. Data shown as mean ± SEM, n=4; *p<0.05, **p<0.01, using 1-way ANOVA. C) ATP viability assay with E3330 in Jurkat cells overexpressing Bcl-2 (Jurkat/Bcl2) in comparison to control, vector-expressing cells (Jurkat/Neo); analyses at 96hrs. Data shown as mean ± SEM, from 4 independent experiments; *p<0.05, **p<0.01, for Jurkat/Bcl2 vs. Jurkat/Neo, using 2-way ANOVA.

Article Snippet: The Jurkat/Bcl2 and Jurkat/Neo are sublines derived from transfection of Jurkat cells respectively with a psFFV-neo expressing vector containing human BCL2 or empty vector, and were obtained from ATCC in 2014.

Techniques: Expressing, Control, Viability Assay, Comparison, Plasmid Preparation

Journal: Scientific Reports

Article Title: 6E11, a highly selective inhibitor of Receptor-Interacting Protein Kinase 1, protects cells against cold hypoxia-reoxygenation injury

doi: 10.1038/s41598-017-12788-4

Figure Lengend Snippet: Characterization of hit compound 6E11 as new necroptosis inhibitor. ( a ) Workflow of the cell-based screening of ICBMS chemical library for the selection of new inhibitors of necroptosis. Among 2,800 compounds, 6E11, was selected as the more potent inhibitor of TNF-α-induced necroptosis in human FADD-deficient Jurkat T cells. The chemical structures of 6E11 and its negative control (8A03) are depicted above the workflow. The primary screening is performed in monoplicate. The negative control was not detected during the screening campaign. ( b ) Dose-dependent protection of 6E11 against TNF-α-induced Jurkat FADD deficient cell necroptosis. After a 24-h incubation of the cells with or without (w/o) TNF-α and increasing concentrations of tested compounds, the effect on the cell viability was evaluated by MTS reduction assay. The cells were treated only with the tested compound to evaluate its putative toxicity. The values were normalized as a percentage of cell viability, considering 100% viable cells in the control treated with DMSO (n = 3, mean ± SD).

Article Snippet: Jurkat wild-type A3, FADD-deficient Jurkat I 2.1 and RPE-1 hTERT human cell lines were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA).

Techniques: Selection, Negative Control, Incubation, Control

Journal: Scientific Reports

Article Title: 6E11, a highly selective inhibitor of Receptor-Interacting Protein Kinase 1, protects cells against cold hypoxia-reoxygenation injury

doi: 10.1038/s41598-017-12788-4

Figure Lengend Snippet: 6E11 inhibits death receptor-induced necroptosis, but not apoptosis. ( a ) Human FADD-deficient Jurkat T cells were treated or not with TNF-α (10 ng/ml) in presence or not of increasing concentrations of 6E11 (0, 1, 5, 10 µM) or Nec-1 (0, 5, 10 µM) for 18 hours. Intracellular ATP levels were measured with the CellTiter-Glo® Luminescent Cell Viability Assay (n = 3, mean ± SEM, * P < 0.05) (EC 50 ~6 µM). ( b ) Human FADD-deficient Jurkat T cells were treated or not with TNF-α (10 ng/ml) in presence or not of increasing concentrations of 6E11 (0, 5, 10, 20 µM) or Nec-1 (0, 5, 10 µM) for 18 hours. The percentage of cell death was determined by propidium iodide staining using flow cytometry (n = 3, mean ± SEM, ** P < 0.01 and *** P < 0.001). ( c ) Human FADD-deficient Jurkat T cells were treated or not with TNF-α (10 ng/ml) in presence or not of increasing concentrations of 6E11 (0, 5, 10, 20 µM) for 18 hours. The mitochondrial transmembrane potential (MTP) was measured using the fluorescent dye DiOC6 (3) and flow cytometry analysis. ( d ) 6E11 inhibits TRAIL-induced necroptosis. Wild-type Jurkat T cells were treated or not with TRAIL (10 ng/ml), Z-VAD (30 µM) and CHX (1 µg/ml) for 18 hours in presence or not of increasing concentrations of 6E11 (0, 5, 10, 20 µM) or 10 µM Nec-1. The percentage of cell death was determined by propidium iodide staining using flow cytometry (n = 3, mean ± SEM, ** P < 0.01). ( e ) 6E11 does not inhibit TRAIL-induced apoptosis. Wild-type Jurkat T cells were treated or not with TRAIL (20 ng/ml) for 18 hours in presence or not of increasing concentrations of 6E11 (0, 5, 10, 20 µM) or Nec-1 (0, 5, 10, 20 µM). The percentage of cell death was determined by propidium iodide staining using flow cytometry (n = 3, mean ± SEM).

Article Snippet: Jurkat wild-type A3, FADD-deficient Jurkat I 2.1 and RPE-1 hTERT human cell lines were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA).

Techniques: Cell Viability Assay, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: 6E11, a highly selective inhibitor of Receptor-Interacting Protein Kinase 1, protects cells against cold hypoxia-reoxygenation injury

doi: 10.1038/s41598-017-12788-4

Figure Lengend Snippet: 6E11 is not cytotoxic at inhibitory concentration and protects from necroptosis even after cell death initiation. ( a ) 24 hours after treatment with increasing concentrations of 6E11, cell viability was measured with a MTS assay to determine the toxicity of the compound towards either human PBL (upper panel) (n = 6 individuals, mean ± SEM) or human retina pigmented epithelial cells (RPE-1 hTERT) (lower panel) (n = 3, mean ± SD). ( b ) Viability was measured with a MTS assay in FADD-def Jurkat cells treated with TNF-α (10 ng/ml) followed by 10 µM 6E11 addition 1 to 4 h post necroptosis initiation (n = 4, mean ± SEM, * P < 0.05; ** P < 0.01).

Article Snippet: Jurkat wild-type A3, FADD-deficient Jurkat I 2.1 and RPE-1 hTERT human cell lines were obtained from ATCC (American Type Culture Collection, Rockville, MD, USA).

Techniques: Concentration Assay, MTS Assay