Project description:We used microarray to monitor the differentially expresed genes during Jurkat T cell activaiton. Jurkat T cells with control shRNA or IKKe shRNA were treated with solvent or PMA and ionomycin for 3 h, and then RNA was extracted and applied to microarray analysis
Project description:Aim: to perform a genome-wide investigation of chromatin landscape and gene expression patterns downstream of calcium and kinase signaling in Jurkat T cells. Methods: PMA and ionomycin were used to activate the calcium and kinase signalling networks involved in T cell activation. Global gene expression was measured using RNA-seq, whilst ATAC-seq was used to probe chromatin landscape following 3 hours of stimulation with PMA, ionomycin or both. All experiments were performed in triplicate. For RNA-seq all sequencing was performed using paired-end sequencing on an Illumina HiSeq2500 instrument. For ATAC-seq sequencing was performed using a HiSeq 1500. Results: we mapped approximately 60 million reads per sample for ATAC-seq, and 22 million reads per library for RNA-seq. Overall we identified 57,825 transcripts and 19,763 ATAC-seq peaks. We identifiead 1648 genes whose expression was increased by 2-fold or more by at least one treatment in comparison to untreated cells. Similarly, we identified 3972 ATAC peaks that were induced by at least 2-fold by treatment in comparison to untreated cells. Conclusions: we found that chromatin landscape was associated with gene expression downstream of calcium and kinase signaling in Jurkat cells. Further to this we found that activation of the full complement of TCR-responsive genes is dependent upon both PMA and ionomycin, and amounts to more than just the sum of both.
Project description:To assess the effects of small molecules SP100030 and Selinexor on Jurkat T cells activated by PMA/Ionomycin after 6 hours of activation and treatment
Project description:Purpose: study the role of MALT1 auto-proteolysis in T cell receptor mediated activation of NF-kB. Methods: Jurkat cells were generated that express wild type MALT1, the auto-cleavage deficient MALT1-R149A mutant, the catalytic inactive MALT1-C464A mutant or the R149A-C464A double mutant (RACA). Expression of endogenous MALT1 was inactivated using TALEN technology for the Jurkat cells expressing MALT1-R149A (JDM-RA) and MALT1-C464A (JDM-CA). Illumina HISeq 2000 deep sequencing was performed to determine the mRNA profiles for MALT1, JDM-RA, JDM-CA and RACA cells in unstimulated conditions or after treatment with 75ng/ml PMA and 150 ng/ml ionomycin for 3 or 18 hrs. Results: PMA ionomycin stimulation of the MALT1 auto-cleavage defective JDM-RA cells fails to activate NF-kB-dependent transcription like for the MALT1 catalytic inactive JDM-CA cells and the double RACA mutant cells. Conclusion: MALT1 autoproteolysis is essential for transcription of NF-kB target genes
Project description:In order to determine the calcineurin inhibitory effect of CABIN1 peptide, we performed RNA-sequencing in Jurkat T cells expressing negative contorl (HA-mCherry) or HA-mCherry-CABIN1 peptide. Jurkat T cells were activated by treatment 40 nM PMA and 1 μM Ionomycin for 8 hr. 0.5 μM FK506 (Tacrolimus, Tac) was pretreated for 1 hr before treatment with PMA and Ionomycin. Total RNA was extracted from these cells. Extracted RNA was used to prepare an mRNA sequencing library using TruSeq Stranded mRNA sample preparation kit. All samples were sequenced on Illumina NextSeq 500 with a 75 bp paired end read.
Project description:To investigate the cooperative function USF factors for the regulation of T cell transcriptom, we established Jurkat mHIV-Luciferase cells in which USF1 or USF2 was knocked out by CRISPR-Cas9. We then performed gene expression profiling analysis using data obtained from RNA-seq of cell lines either left untreated or stimulated by PMA and Ionomycin co-treatment.
Project description:To investigate the cooperative function TFII-I and TRIM24 in the regulation of T cell activation regulated genes, we established Jurkat Tat cells in which TFII-I was depleted by shRNA and TRIM24 was knocked out by CRISPR-Cas9. We then performed gene expression profiling analysis using data obtained from RNA-seq of cell lines stimulated by PMA and Ionomycin.
Project description:Jurkat T cells were activated with PMA and ionomycin. Total and Labeled fragmented RNA was extracted every 5 minutes during a time course up to 15min after activation. Biological duplicates of all samples were sequenced on a HiSeq 1500
Project description:A quantitative label-free secretome analysis protocol using a click chemistry-based approach for the enrichment of secreted glycoproteins was adapted for and applied to a T cell model. There, Jurkat cells were activated via PMA/ionomycin and the dynamic modulation of the T cell secretome was investigated and compared to the dynamic modulation of the T cell proteome of the same model.
