human leukemic jurkat e6.1 cell line  (European Collection of Authenticated Cell Cultures)

Journal: bioRxiv

Article Title: Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters

doi: 10.1101/617126

Figure Lengend Snippet: A) Jurkat T cells stably transduced with mYFP.NEMO were activated on stimulatory glass surfaces coated with 10 μg/ml anti-CD3 (OKT3), 1 μg/ml rhVCAM1 and/or 10 μg/ml fibronectin, as indicated. Dotted blue arrows, marked ‘P’, identify perinuclear pools of NEMO. Objects marked with a red ‘M’ are typical of the large objects referred to as ‘macroclusters’. Similar patterns were observed in every experiment (n≥3 for each condition). Scale bars: 10 μm. B) Primary human T cell blasts were transduced, stimulated, and imaged as in A. Images are representative of four experiments. C) The lag between local contact initiation and NEMO microcluster formation was calculated for cells captured in the process of spreading. Data are presented as the mean ± SD based on the number of cells examined. 468 clusters were observed in thirteen cells stimulated on anti-CD3 (3, n=8 cells) or anti-CD3 and rhVCAM1 (3-V, n=2 cells). No significant differences were observed between any group of cells and the pool of all conditions, or the cells stimulated on anti-CD3. D) Jurkat T cells stably transduced with mYFP.NEMO were stimulated on anti-CD3 and rhVCAM1 and fixed after five minutes. NEMO clusters were identified algorithmically (see Materials and Methods). The resulting masks are pseudo-colored (green) and superimposed on the raw image (red). Line scans show NEMO intensity (red line) along the indicated white line relative to the cluster masks (shaded green). E) Jurkat T cells were transiently transfected or stably transduced with mYFP.NEMO and imaged on the indicated substrates. NEMO clusters identified as in D were binned into classes based on cluster area. Classes are labeled using the diameter of a circle with an area equivalent to the upper bound of the class. Graphs present the cluster count (upper), the fractional distribution of clusters by class (middle), and the fraction of the NEMO intensity in the imaging plane that is captured within each class (lower). Data are presented as the mean ± SEM, based on the number of cells analyzed. Cluster quantitation was performed using 23 cells stimulated on anti-CD3 and rhVCAM1 and 13 cells stimulated on rhVCAM1 alone. Statistical differences among classes were determined using Student’s T-test: p < 0.05, *; p < 0.01, **; p < 0.001, ***. F) Jurkat cells expressing mYFP.NEMO and NFκB-luciferase plus either mCer3 itself or mCer3 chimeras with WT or kinase-deficient (KR) IKKβ were stimulated as indicated; levels of luciferase were measured to indicate the amount of NF-κB transcription. G) Jurkat T cells stably expressing mYFP.NEMO WT and either mCer3.IKKβ WT or mCer3.IKKβ-K44R (kinase-deficient mutant). Kymographs for each were taken from the region in the white box, and represent 5 minutes in time. H) Model showing the location of NEMO S68 in the context of a NEMO dimer; phosphorylation of this site is predicted to cause destabilization of the IKK complex and dissolution of the NEMO/ IKKβ microcluster.

Article Snippet: The Jurkat human leukemic T cell line E6.1 (TIB-152; RRID:CVCL_0367) and the C305 hybridoma (CRL-2424; RRID:CVCL_K130) were obtained from ATCC (Manassas, VA).

Techniques: Stable Transfection, Transduction, Transfection, Labeling, Imaging, Quantitation Assay, Expressing, Luciferase, Mutagenesis, Phospho-proteomics, Dissolution

Journal: bioRxiv

Article Title: Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters

doi: 10.1101/617126

Figure Lengend Snippet: A) SLP76-deficient J14 Jurkat cells stably reconstituted with SLP-76.YFP, parental Jurkat T cells and primary human T cell blasts were transiently transfected as indicated and stimulated on anti-CD3, rhVCAM1, and anti-CD28, as in . Images were pseudo-colored as indicated. The regions enclosed in magenta boxes are enlarged at right. Relative fluorescence intensities along the white diagonal lines are shown in the lower panels. Data are representative of three-six experiments for Jurkat cells and two experiments for primary cells. B) Jurkat T cells were transiently transfected with mYFP.NEMO and ZAP-70.mCFP, stimulated on anti-CD3, and imaged over time. A pseudo-colored still image is shown at left. Blue arrows identify a perinuclear pool that is not anchored to the substrate. The regions enclosed in magenta boxes were used to generate the kymographs shown at right. White arrows identify points at which static NEMO clusters begin to move, and asterisks identify the points at which mobile NEMO clusters stop. Still images are representative of six experiments. Scale bars: 10 μm (stills); 2 μm (insets); 5 μm × 60 seconds (kymographs). C) Graphs present the fractional distribution of NEMO clusters by size class (left), the fraction of total ZAP intensity within each NEMO class (center-left), the fraction of the total area masked as a ZAP cluster that lies within each NEMO class (center), the per-pixel enrichment of ZAP intensity within each NEMO class, relative to the entire cell (center-right), and the per-pixel enrichment of ZAP masked area within each NEMO class, relative to the entire cell (right). Statistical differences among corresponding classes were determined using Student’s T-test: p < 0.05, *; p < 0.01, **; p < 0.001, ***. Data are presented as the mean ±SEM, based on the number of cells analyzed. Calculations were performed for 12 cells co-expressing NEMO and ZAP-70.

Article Snippet: The Jurkat human leukemic T cell line E6.1 (TIB-152; RRID:CVCL_0367) and the C305 hybridoma (CRL-2424; RRID:CVCL_K130) were obtained from ATCC (Manassas, VA).

Techniques: Stable Transfection, Transfection, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters

doi: 10.1101/617126

Figure Lengend Snippet: A) Diagram of NEMO domain structure and truncations used in subsequent experiments. B) NEMO-deficient 8321 cells expressing an NF-κB-driven rat Thy1 reporter were transiently transfected with vectors expressing mYFP alone, or mYFP.NEMO chimeras bearing the indicated deletions. After stimulation with PMA, plus either ionomycin or anti-CD28, these cells were analyzed by flow cytometry. Thy1 MFI is shown for cells expressing comparable levels of mYFP. Statistical differences were determined using Student’s T-test: p < 0.01, **; p < 0.001, ***. Data are presented as the mean ± SD of three technical replicates and are representative of 2-5 experiments. C) Jurkat T cells stably expressing wild-type or truncated mYFP.NEMO chimeras were stimulated as in and fixed after five minutes. Still images are representative of the cells quantitated in panel D. Scale bars: 10 μm. D) Transiently transfected and stably transduced Jurkat T cells expressing the indicated constructs were stimulated on anti-CD3 and rhVCAM1 and imaged live or after fixation. NEMO clusters were analyzed as in . Data are presented as the mean ± SEM, based on the number of cells analyzed. Numbers in parentheses indicate the number of cells analyzed per condition; numbers in brackets indicate the number of independent experiments from which these cells were derived. Statistical differences among corresponding classes were determined using Student’s T-test: p < 0.05, *; p < 0.01, **; p < 0.001, ***.

Article Snippet: The Jurkat human leukemic T cell line E6.1 (TIB-152; RRID:CVCL_0367) and the C305 hybridoma (CRL-2424; RRID:CVCL_K130) were obtained from ATCC (Manassas, VA).

Techniques: Expressing, Transfection, Flow Cytometry, Stable Transfection, Construct, Derivative Assay

Journal: bioRxiv

Article Title: Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters

doi: 10.1101/617126

Figure Lengend Snippet: A) Diagram of NEMO point mutations used in subsequent experiments. B) The functionality of mYFP.NEMO chimeras bearing mutations that impair poly-ubiquitin binding was assessed as in . Data are presented as mean ± SD of 3 technical replicates and are representative of 3-5 experiments. C) Jurkat T cells stably expressing wild-type or mutant mYFP.NEMO chimeras were stimulated on anti-CD3 and rhVCAM1-coated substrates and imaged continuously for at least 5 minutes. Still images are representative of the cells quantitated in panel D. Kymographs were derived from the sub-regions boxed in red. Scale bars: 10 μm. D) Jurkat T cells expressing the indicated constructs were stimulated on anti-CD3 and rhVCAM1. NEMO clusters were imaged, analyzed, and presented as in . E) Primary human T cell blasts were transfected with vectors encoding either a wild-type or a Y301S mutant mYFP.NEMO chimera and a ZAP-70.TRT chimera. Transfected cells were stimulated on anti-CD3, rhVCAM1, and anti-CD28, fixed after 5 minutes, and imaged as above. Images are representative of 4 experiments for NEMO.WT and 2 experiments for NEMO.Y301S. Scale bars: 10 μm.

Article Snippet: The Jurkat human leukemic T cell line E6.1 (TIB-152; RRID:CVCL_0367) and the C305 hybridoma (CRL-2424; RRID:CVCL_K130) were obtained from ATCC (Manassas, VA).

Techniques: Ubiquitin Proteomics, Binding Assay, Stable Transfection, Expressing, Mutagenesis, Derivative Assay, Construct, Transfection

Journal: bioRxiv

Article Title: Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters

doi: 10.1101/617126

Figure Lengend Snippet: A) Lck-deficient J.CaM1.6 Jurkat cells were transiently transfected with vectors encoding either mYFP.NEMO alone (left) or mYFP.NEMO and wild-type Lck.Myc.TRT (right), stimulated on substrates coated with anti-CD3 and rhVCAM1, and fixed after 5 minutes. Still images are representative of the cells examined in panel B. B) Jurkat T cells stably expressing wild-type mYFP.NEMO were pre-incubated with 10μM PP2 or a corresponding volume of DMSO for 10 minutes. Cells were stimulated on surfaces coated with anti-CD3 and rhVCAM1 in the presence of the corresponding compounds. Images were acquired continuously for five minutes, beginning two and ten minutes after the addition of cells. Still images derived from the later series are representative of the cells quantitated in panel C. Kymographs derived from both acquisitions are shown. The later kymograph is derived from the region boxed in red. C) Jurkat T cells stably expressing wild-type mYFP.NEMO were pre-treated with 10μM PP2, 100μM piceatannol, or independent DMSO controls, and stimulated on anti-CD3 and rhVCAM1. NEMO clusters were imaged and analyzed as in . D) ZAP-70-deficient P116 Jurkat T cells were transiently transfected with vectors encoding mYFP.NEMO and either a wild-type (WT) or a kinase-dead (K369R, KR) ZAP-70.mCFP chimera. Cells were stimulated on substrates coated with anti-CD3, rhVCAM1, and anti-CD28 and imaged continuously for five minutes. Still images are shown above. Kymographs are derived from the regions boxed in white. Images are representative of two experiments. Scale bars: 10 μm (stills); 5 μm × 60 seconds (kymographs). See for NEMO cluster quantitation in J.CaM1.6 and P116 cells.

Article Snippet: The Jurkat human leukemic T cell line E6.1 (TIB-152; RRID:CVCL_0367) and the C305 hybridoma (CRL-2424; RRID:CVCL_K130) were obtained from ATCC (Manassas, VA).

Techniques: Transfection, Stable Transfection, Expressing, Incubation, Derivative Assay, Quantitation Assay

Journal: bioRxiv

Article Title: Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters

doi: 10.1101/617126

Figure Lengend Snippet: A) Wild-type Jurkat T cells and mutant lines lacking LAT (J.CaM2.5), SLP-76 (J14) or Carma1 (JPM50.6) were stably transduced with wild-type mYFP.NEMO, stimulated on anti-CD3 and rhVCAM1, and fixed after five minutes. Still images are representative of the cells examined in panel B. B) Mutant cell lines were prepared and stimulated as in A. NEMO clusters were imaged and analyzed as in . C-D) JPM50.6 Jurkat T cells were transiently transfected with vectors expressing fluorescent chimeras of NEMO and Zap70, and then stimulated, fixed, and imaged as in A. Representative still images and kymographs are shown (n=9 cells in 2 experiments). C) JPM50.6 cells were transfected with vectors expressing mYFP.NEMO and Zap70.mRFP1. Images were acquired continuously for five minutes, beginning two or 20 minutes after the addition of cells. Still images acquired early in each series are shown at left, with kymographs derived from the boxed regions at right (n=4 cells in 2 experiments for each condition). Scale bars: 10 μm (stills); 5 μm × 60 seconds (kymographs).

Article Snippet: The Jurkat human leukemic T cell line E6.1 (TIB-152; RRID:CVCL_0367) and the C305 hybridoma (CRL-2424; RRID:CVCL_K130) were obtained from ATCC (Manassas, VA).

Techniques: Mutagenesis, Stable Transfection, Transduction, Transfection, Expressing, Derivative Assay

Journal: Clinical and Experimental Immunology

Article Title: Cultured lymphocytes’ mitochondrial genome integrity is not altered by cladribine

doi: 10.1093/cei/uxad112

Figure Lengend Snippet: Proliferation rates of the T-cell lines and effects of cladribine on cell viability. (a) The growth rate of Jurkat E6.1 is faster than the CCRF-CEM cell line ( n = 3). (b) A cladribine dose response curve in Jurkat E6.1 cell line for cell proliferation with 48 hours ( n = 5) and 96 hours culture ( n = 5). (c) The effect of cladribine on cell proliferation in Jurkat with 12 days ( n = 5) and 16 days in culture ( n = 5). (d) The effect of cladribine on cell proliferation in CCRF-CEM with 2 days ( n = 5), 8 days ( n = 4), and 12 days ( n = 4) in culture ( n = 5).

Article Snippet: The human leukemic Jurkat E6.1 cell line (pseudodiploid, modal No 46) and the CCRF-CEM (ECACC 85112105) cell line (2 n = 46) were obtained from the European Collection of Authenticated Cell Cultures (ECACC) general cell collection and cultured according to the recommended instructions (Jurkat E6.1: RPMI 1640; 2 mM glutamine; 10% FBS) (CCRF-CEM: RPMI 1640; 2 mM glutamine; 20% FBS).

Techniques:

Journal: Clinical and Experimental Immunology

Article Title: Cultured lymphocytes’ mitochondrial genome integrity is not altered by cladribine

doi: 10.1093/cei/uxad112

Figure Lengend Snippet: Effect of cladribine on mitochondrial protein synthesis. (a) Immunoblot of MT-ATP6, MT-CO1, MRPL11, MRPS35, CASPASE3, SDHA, and CANX after 12 days culture with cladribine. In this experiment, 10 nM cladribine in CCRF cells resulted in extensive cell death, and immunoblot was not performed. (b) A pulse metabolic labeling of mitochondrial protein synthesis. The effect on mitochondrial protein synthesis in the CCRF-CEM and Jurkat cells after treatment with cladribine for 8 days ( n = 3) or 12 days ( n = 3).

Article Snippet: The human leukemic Jurkat E6.1 cell line (pseudodiploid, modal No 46) and the CCRF-CEM (ECACC 85112105) cell line (2 n = 46) were obtained from the European Collection of Authenticated Cell Cultures (ECACC) general cell collection and cultured according to the recommended instructions (Jurkat E6.1: RPMI 1640; 2 mM glutamine; 10% FBS) (CCRF-CEM: RPMI 1640; 2 mM glutamine; 20% FBS).

Techniques: Western Blot, Labeling