cell lines cell line source s human acute leukemic jurkat t cells  (ATCC)

Journal: Chemical Science

Article Title: Direct decarboxylation of ten-eleven translocation-produced 5-carboxylcytosine in mammalian genomes forms a new mechanism for active DNA demethylation †

doi: 10.1039/d1sc02161c

Figure Lengend Snippet: Decarboxylation of F-5caC is a rapid process and occurs in diverse mammalian cells. (a–c) Quantification of F-5caC, F-dC, and F-5mC levels in genomic DNA of HEK293T cells upon feeding of 300 μM of F-5caC for different times. (d) Quantification of F-5caC, F-dC, and F-5mC levels in genomic DNA of Jurkat-T, MCF-7, and Neuro-2a cells upon feeding of 300 μM of F-5caC for 3 d. (e) Schematic illustration of the active 5mC demethylation through two pathways: the direct decarboxylation of TET-produced 5caC to cytosine in mammalian genomes established in the current study and the previous TET-TDG-BER pathway.

Article Snippet: The human embryonic kidney epithelial cell line (HEK293T), human leukemic cell line (Jurkat-T), human breast adenocarcinoma cell line (MCF-7), and mouse neuroblastoma N2a cell line (Neuro-2a) were obtained from the China Center for Type Culture Collection (Wuhan, China).

Techniques: Produced

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: Oncotarget

Article Title: PD-L1 checkpoint blockade delivered by retroviral replicating vector confers anti-tumor efficacy in murine tumor models

doi: 10.18632/oncotarget.26785

Figure Lengend Snippet: Mixtures of SEE loaded, SEE + anti-human PD-L1 (10 µg/mL) or SEE + purified scFv-PD-L1 (10 µg/mL) Raji B cells or Raji B cells overexpressing human PD-L1 (with rested Jurkat T cells overexpressing human PD-1) were co-cultured in RPMI-1640 medium containing 10% human AB serum for stimulation for 20–22 hours followed by IL-2 measurement by ELISA.

Article Snippet: Human leukemic T cell line Jurkat Clone E6-1 (ATCC, TIB-152) and human Burkitt’s B lymphoma cell line Raji (ATCC, CCL86) were cultured in complete RPMI-1640 medium.

Techniques: Purification, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Calreticulin and integrin alpha dissociation induces anti-inflammatory programming in animal models of inflammatory bowel disease

doi: 10.1038/s41467-018-04420-4

Figure Lengend Snippet: ER-464195-01 inhibits interaction of CRT with ITGA. a In situ PLA assay for the interaction of CRT with ITGA4 at the mucosa (upper) and the vascular lumen (bottom) in the UC colon. Representative images; non-inflamed control (left) and the inflamed site (right). Right two panels of each site (control or inflamed); enlarged boxes of left two panels in each site. Scale bars, 20 μm. b Quantitative analysis of a . Results are given as the mean ± SEM of n = 3; * P < 0.05 (two-tailed t test), the inflamed (Inf) group versus non-inflamed control (Con) group. c Chemical structures of small molecules, ER-464195-01, ER-435813-01 and ER-339093-13. d IC 50 values for inhibition of CRT binding to three types of ITGA peptides. Results are given as the mean ± SEM of n = 3 independent experiments performed in duplicate. e Expansion of two-dimensional 15 N-HSQC spectra of the uniformly 15 N-labeled intracellular domain of ITGA4 (black), with the CRT (red), and with combination of CRT and ER-339093-13 (green). An overall view of the spectra is shown in Supplementary Fig. . f 1 H 1D NMR spectra of ER-339093-13 or ER-435813-01 with/without the CRT. Data are representative of at least two independent experiments. g In vitro pull-down assay, a dose-dependent-specific inhibition of GST-fused CRT proteins binding to ITGA4 by ER-464195-01 or ER-435813-01. h In situ PLA assay for CRT–ITGA4 interaction (red dots) in PMA (100 ng/mL, 30 min)-stimulated Jurkat cells with 5 μM of ER-464195-01 or ER-435813-01. Hoechst 33258 staining (blue); the nucleus. Scale bars, 20 µm. i Quantitative analysis of h , five independent experiments with at least 500 cells scored in each experiment. Results are given as mean ± SEM of n = 5; ** P < 0.01 and *** P < 0.0001 and ns (not significant). Statistical significance was evaluated using one-way ANOVA with Bonferroni’s multiple comparison test

Article Snippet: Human leukemic T lymphoblast Jurkat cell line was purchased from RIKEN Cell Bank and Dainippon Pharmaceutical Co., Ltd.

Techniques: In Situ, Control, Two Tailed Test, Inhibition, Binding Assay, Labeling, In Vitro, Pull Down Assay, Staining, Comparison

Journal: Nature Communications

Article Title: Calreticulin and integrin alpha dissociation induces anti-inflammatory programming in animal models of inflammatory bowel disease

doi: 10.1038/s41467-018-04420-4

Figure Lengend Snippet: ER-464195-01 suppresses the cell adhesion and the migration of leukocytes. a , b Inhibition of MnCl 2 -induced T cells and neutrophils adhere to VCAM-1 a and ICAM-1 b , respectively, by ER-464195-01 or ER-435813-01. Results are given as the mean ± SEM of n = 3 independent experiments performed in duplicate. c , d IC 50 values for inhibition of PMA-induced T cells and fMLP-induced neutrophils adhere to VCAM-1 c and ICAM-1 d , respectively, by ER-464195-01 or ER-435813-01. Results are given as the mean ± SEM of n = 3 independent experiments performed in duplicate. e , Flow cytometric analysis for the cell surface CRT in PMA-stimulated Jurkat cells with ER-464195-01 or ER-435813-01. f , Quantitative analysis of e . Results are given as mean ± SEM of n = 6 experiments; * P < 0.05, ** P < 0.01 and ns (not significant). Statistical significance was evaluated using one-way ANOVA with Bonferroni’s multiple comparison test. MFI, mean fluorescence intensity. g , h In vivo efficacy of ER-464195-01 on OXA-induced lymph cells g and glycogen-induced PECs h infiltration into OXA-treated colons. Results are given as the mean ± SEM of n = 6 or 7; # P < 0.0001, OXA group versus control (without the injection of OXA emulsion) (two-tailed t test). * P < 0.05, ** P < 0.01, *** P < 0.001 and ns, OXA with ER-464195-01 groups versus OXA group (one-way ANOVA followed by Dunnett’s multiple comparison test)

Article Snippet: Human leukemic T lymphoblast Jurkat cell line was purchased from RIKEN Cell Bank and Dainippon Pharmaceutical Co., Ltd.

Techniques: Migration, Inhibition, Comparison, Fluorescence, In Vivo, Control, Injection, Emulsion, Two Tailed Test

Journal: BioMed Research International

Article Title: MicroRNA Expression Profiling in Psoriatic Arthritis

doi: 10.1155/2018/7305380

Figure Lengend Snippet: Validation of differentially expressed miRNAs by real-time PCR. (a) Real-time PCR for the indicated miRNAs were performed in healthy controls (Healthy), PsA active, and nonactive samples included in the microarray and in 7 patients affected by RA. (b) Real-time PCR for miR-126-3p in an expanded cohort of healthy (15 patients), PsA active (11 patients), and PsA nonactive (12 patients) groups. Values are calculated with ΔCt method. miR-16-5p and miR-26a-5p were used as endogenous controls for miRNA expression (see Methods) . Histograms indicate mean values; bars indicate standard deviation (SD). ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001 (Mann–Whitney rank sum test). (c) Real-time PCR expression of miR-126-3p in Jurkat cells after 48 h from transfection with miRNA mimics. miR-16-5p and miR-26a-5p were used as endogenous controls. (d) Identification of novel miR-126-3p targets. Real-time PCR expression of the indicated transcripts in mimic miR-126-3p-transfected and control cells. Beta-Actin was used as endogenous control. Values were calculated with ΔΔCt method as fold change with respect to mimic negative control-transfected samples. All the histograms indicate mean values of at least three independent experiments; bars indicate standard deviation (SD). ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001 (Student's t -test).

Article Snippet: Human leukemic T-cell lymphoblast cell line Jurkat was purchased from European Collection of Authenticated Cell Cultures (clone E6.1, ECACC 88042803).

Techniques: Biomarker Discovery, Real-time Polymerase Chain Reaction, Microarray, Expressing, Standard Deviation, MANN-WHITNEY, Transfection, Control, Negative Control