human acute t cell leukemia line jurkat  (ATCC)

Journal: Epigenomes

Article Title: Genome-Wide Methylation Profiling of Peripheral T–Cell Lymphomas Identifies TRIP13 as a Critical Driver of Tumor Proliferation and Survival

doi: 10.3390/epigenomes8030032

Figure Lengend Snippet: TRIP13 is frequently upregulated in primary human PTCL. ( A ) Relative TRIP13 expression as determined by RNA-seq. Data are presented as fold differences in FPKM values relative to the mean of normal T–cells (C; n = 4) obtained by RNA-seq analysis. Statistically significant differences are indicated by *. ( B ) TRIP13 expression as determined by RNA-seq analysis of normal human T–cells (C, n = 4), two sets of human anaplastic large cell lymphomas (ALCL-1, n = 5 tumors T1-T5 profiled in this study; ALCL-2, n = 21 publicly available data), natural killer T–cell lymphomas (NKTCL, n = 15), adult T–cell leukemia/lymphomas (ATLL, n = 8), T-lymphoblastic lymphomas (TLBL, n = 8) and PTCL-NOS ( n = 5, tumors T6-T10 profiled in this study). The horizontal line represents the median, bounds of the box-like range of variation, and whiskers min and max values. Pairwise comparisons between each tumor group and controls are statistically significant ( p < 0.05 by DESeq2) indicated by *. ( C ) Immunoblot analysis of TRIP13 protein levels in human lymphomas and leukemia cell lines. T8ML-1 (PTCL-NOS); JURKAT (acute T–cell leukemia); SUP-T1 (T–cell lymphoblastic lymphoma); HH (cutaneous T–cell leukemia/lymphoma), MEC1, MEC2 (chronic B-cell leukemia); RAJI (Burkitt’s lymphoma); K-562 (chronic myelogenous leukemia); LOUCY, CCRF-CEM (acute T–cell lymphoblastic leukemia); Mo T (hairy T–cell leukemia); MJ (cutaneous T–cell lymphoma); DND41 (acute T–cell lymphoblastic leukemia); MOLT4 (T lymphoblast cell); CTV-1 (T-ALL). HSC70 served as a loading control.

Article Snippet: Human acute T–cell leukemia cell line JURKAT (#TIB-152) was obtained from the American Type Culture Collection (ATCC, Gaithersburg, MD, USA).

Techniques: Expressing, RNA Sequencing, Western Blot, Control

Journal: Epigenomes

Article Title: Genome-Wide Methylation Profiling of Peripheral T–Cell Lymphomas Identifies TRIP13 as a Critical Driver of Tumor Proliferation and Survival

doi: 10.3390/epigenomes8030032

Figure Lengend Snippet: TRIP13 downregulation results in impaired cellular proliferation of malignant T–cells. ( A ) Representative examples of FACS diagrams indicating mCherry expression measured in unselected T8ML-1 cell line transduced with lentiviruses expressing shRNA against either scrambled (scr) or TRIP13 (shRNA-1). Data obtained at days 2 and 18 upon continuous culturing in vitro are shown. The percentage of mCherry positive cells is shown above the gated population. ( B ) Percentage of mCherry-positive cells expressing shRNA-1 against either scrambled or TRIP13 upon continuous culturing in vitro at indicated times as measured by FACS. Data are presented as mean ± SEM (from three independent experiments) relative to scrambled, p < 0.05. p values were calculated by a two-tailed Student’s t -test. ( C ) Relative mCherry expression in T8ML-1 cells transduced with lentivirus encoding shRNA-1 against TRIP13 or scrambled, measured by FACS 48 h after transduction (day 2) and 20 days later upon continuous culturing in vitro. The transduction efficiency, as measured by mCherry expression at day 2 was set to 100%, for both cells transduced with scrambled and TRIP13 shRNAs. The values obtained for the percentage of mCherry-positive cells at each time point were plotted relative to the percentage at day 2. Data are presented as mean ±SEM (from three independent experiments) relative to scrambled, p < 0.05. p values were calculated by a two-tailed Student’s t -test. ( D ) Immunoblot analysis of TRIP13 protein levels in JURKAT cells transduced with lentiviruses expressing either scrambled or TRIP13 shRNAs. Cells were harvested and analyzed 72 h after transduction. HSC70 served as a loading control. ( E ) Relative percentage of live unselected JURKAT cells transduced with the indicated lentiviruses at 99% efficiency as measured by FACS analysis of mCherry expression coexpressed from the lentiviral constructs. Viability was determined as the percentage of cells in the “live gate” in forward scatter FACS diagrams at indicated times. The values obtained for the percentage of mCherry-positive cells at each time point were plotted relative to the percentage at day 2. ( F ) Representative examples of FACS diagrams obtained from BrdU incorporation assays of JURKAT cells transduced with lentivirus expressing shRNA against either scrambled or TRIP13 as determined at 4 and 5 after transduction. Cells were exposed to BrdU for 60 min, harvested and FACS analysis was used to evaluate the percentage of cells that incorporated BrdU. The percentage of positive cells in the FACS profile is shown within each plot. A representative example of two independent experiments is shown. ( G ) Representative examples of FACS diagrams obtained from cell cycle analysis of JURKAT cells transduced with lentivirus expressing shRNA against either scrambled or TRIP13 as determined at 4 and 5 days after transduction. A combination of BrdU incorporation assay and staining with 7-ADD followed by FACS was used. ( H ) Percentage of cells in various phases of the cell cycle in JURKAT cells transduced with lentiviruses expressing shRNA against either scrambled or TRIP13 , determined 4 and 5 days by BrdU incorporation assay coupled with 7-AAD staining and FACS-based analysis. ( I ) Representative examples of FACS diagrams obtained from Annexin V assays of JURKAT cells transduced with lentivirus expressing shRNA against either scrambled or TRIP13 as determined at 4 and 5 days after transduction. The percentage of positive cells in the FACS profile is shown within each plot and indicates ongoing apoptosis.

Article Snippet: Human acute T–cell leukemia cell line JURKAT (#TIB-152) was obtained from the American Type Culture Collection (ATCC, Gaithersburg, MD, USA).

Techniques: Expressing, Transduction, shRNA, In Vitro, Two Tailed Test, Western Blot, Control, Construct, BrdU Incorporation Assay, Cell Cycle Assay, Staining

Journal: Viruses

Article Title: Alphaherpesvirus gB Homologs Are Targeted to Extracellular Vesicles, but They Differentially Affect MHC Class II Molecules

doi: 10.3390/v12040429

Figure Lengend Snippet: Construction of stable cell lines expressing bovine herpesvirus 1 (BoHV-1), pseudorabies virus (PRV), or herpes simplex 1 (HSV-1) glycoprotein B (gB), and characterization of size exclusion chromatography (SEC)-isolated extracellular vesicles (EVs). ( A ) The list of constructed cell lines expressing individual viral gB homologs. Human melanoma Mel JuSo (MJS) and Madin–Darby bovine kidney (MDBK) produce endogenous major histocompatibility (MHC) class II. The Human Jurkat T cell line and swine kidney SK6 cells are MHC II-negative. ( B – E ) Immunoblotting detection of EVs markers Alix and flotillin-2 (flot-2) in cell lysates (cl) of constructed cell lines or SEC-isolated EVs. Calnexin (CNX) and mitochondrial Tom 40 were used as non-EVs control proteins. MJSpuro, MDBKpuro, SK6puro, or untransduced Jurkat cells (Ø) were used as negative controls. ( F ) Representative transmission electron microscopy images of EVs preparations from gB-expressing species-specific cells; scale bar 100 nm.

Article Snippet: Madin–Darby bovine kidney (MDBK) cells (CCL-22, ATCC, Manassas, VA, USA), human melanoma Mel JuSo cells (MJS, a kind gift from Dr. Emmanuel Wiertz, University Medical Center Utrecht, Utrecht, The Netherlands), immortalized porcine alveolar macrophages (PAM, clone 3D4/2, AddexBio, San Diego, CA, USA) and human acute T leukemia cell line Jurkat (E6.1, European Collection of Authenticated Cell Cultures, Salisbury, UK) were cultured in RPMI 1640 (Corning, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific, Waltham, MA, USA) and Antibiotic Antimycotic Solution (Thermo Scientific, Waltham, MA, USA).

Techniques: Stable Transfection, Expressing, Virus, Size-exclusion Chromatography, Isolation, Construct, Western Blot, Control, Transmission Assay, Electron Microscopy

Journal: Viruses

Article Title: Alphaherpesvirus gB Homologs Are Targeted to Extracellular Vesicles, but They Differentially Affect MHC Class II Molecules

doi: 10.3390/v12040429

Figure Lengend Snippet: Immunoblotting detection of BoHV-1 ( A ), PRV ( B ), and HSV-1 gB ( C ) in cell lysates (cl) of constructed cell lines or SEC-isolated EVs. (+): gB-expressing cells; (−): MJSpuro, MDBKpuro, SK6puro, or untransduced Jurkat cells (Ø) were used as negative controls. Virus-infected cell lysates (cl) were analyzed as positive controls; in the case of BoHV-1, gB was immunoprecipitated (IP) with H2 antibody before immunoblotting with anti-BoHV-1 serum. Size markers are in kilodaltons. gBa: uncleaved BoHV-1 and PRV gB precursor; gBb: cleaved N-terminal gB subunit; gBc: cleaved C-terminal gB subunit.

Article Snippet: Madin–Darby bovine kidney (MDBK) cells (CCL-22, ATCC, Manassas, VA, USA), human melanoma Mel JuSo cells (MJS, a kind gift from Dr. Emmanuel Wiertz, University Medical Center Utrecht, Utrecht, The Netherlands), immortalized porcine alveolar macrophages (PAM, clone 3D4/2, AddexBio, San Diego, CA, USA) and human acute T leukemia cell line Jurkat (E6.1, European Collection of Authenticated Cell Cultures, Salisbury, UK) were cultured in RPMI 1640 (Corning, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific, Waltham, MA, USA) and Antibiotic Antimycotic Solution (Thermo Scientific, Waltham, MA, USA).

Techniques: Western Blot, Construct, Isolation, Expressing, Virus, Infection, Immunoprecipitation