ABSTRACT: Nuclear RNA from Raji human B cells was hybridized to NimbleGen arrays to quantify gene expression levels. Three biological replicates, each hybridized to new array.
Project description:Transcription has the capacity to modify mechanically DNA topology, DNA structure, and nucleosome arrangement. Resulting from ongoing transcription, these modifications in turn, may provide instant feedback to the transcription machinery. To substantiate the connection between transcription and DNA dynamics, we charted an ENCODE map of transcription-dependent dynamic supercoiling in human Burkitt lymphoma cells using psoralen photobinding to probe DNA topology in vivo. Dynamic supercoils spread ~1.5 kb upstream of the start sites of active genes. Low and high output promoters handle this torsional stress differently as shown using inhibitors of transcription and topoisomerases, and by chromatin immunoprecipation of RNA polymerase and topoisomerases I and II. Whereas lower outputs are managed adequately by topoisomerase I, high output promoters additionally require topoisomerase II. The genome-wide coupling between transcription and DNA topology emphasizes the importance of dynamic supercoiling for gene regulation. Raji cells: untreated and treated with DRB, CPT and BLAP. Three biological replicates per treatment, each hybridized to new array. Total: 12 samples (4 treatments x 3 replicates).
Project description:Expression of the TM4SF member CD9 on the human Burkitts lymphoma cell line Raji induced increased cell proliferation, motilty and adhesion to fibronectin. CD9 promoted increases in Raji cell proliferation was dependent upon histone deacetyalase (HDAC) activity as treatment with HDAC inhibitors trichostain A or cucurmin attenuated CD9 mediated increases in Raji cell proliferation. Gene expression of Raji cells stably expressing human CD9 via transfection with expression vector PRVCMVCD9 was compared with corresponding Mock transfected cell by microarray analysis using the Affymetrix U133 2.0 platform. Experiment Overall Design: Transfected Raji cells that had been in cultured in RPMI, 10% FBS in the presence of 1mg/ml G418 were harvested during exponential growth and RNA was extracted using TRiReagent according to manufacturer's protocol (Sigma).
Project description:In order to determine changes in phosphorylation events in macrophages upon phagocytosis of cancer cells, mouse macrophages were co-cultured with human Raji cancer cells. Macrophages were collected as 0, 1, 15, 60 and 120 minutes after incubation. Phosphosite abundances were compared between timepoints to determine differential phospho signaling over time.
Project description:Raji cells were transduced to express either that canonical IL-13Rα1 subunit of the shared type II receptor for IL-4 and IL-13 or a newly discovered, termed IL-13Rα1-LOR1a. These cells were treated with IL-13 and compared with untreated control cells, as well as with treated and untreated untransduced Raji cells, with the aim of assessing the potential of the IL-13Rα1-LOR1a isoform to transmit IL-13 signals. Each condition was tested in triplicate.
Project description:(i) RNA transcripts of Raji iBZLF1 cells (https://www.biorxiv.org/content/10.1101/573659v1) were compared between non-induced cells and cells induced with doxycycline (100 ng/ ml) for 6 h. Human (Hg19) as well as viral (EBV, Raji KF717093.1) transcripts were analyzed. Artificial ERCC Spike-ins (Thermo Scientific) were added to selected samples to control for global changes of RNA levels. (ii) For control purposes, the RNA transcripts in Raji cells equipped with a doxycycline-controlled truncated BZLF1 allele lacking its transactivation domain (Raji iBZLF1 AD-truncated) were prior to and after induction for 6 h. (iii) For another control, the RNA transcripts of parental, unmodified Raji cells were analyzed prior to and after doxycycline induction for 6 h. All experiments were performed as triplicates.
Project description:miR-142-3p is highly expressed in peripheral blood mononuclear cells (PBMCs) and has been described as a hematopoietic-restricted lineage, suggesting immune functions (Chen, Li et al. 2004; Landgraf, Rusu et al. 2007; Merkerova, Belickova et al. 2008). In order to determine the roles of miR-142-3p in B lymphocytes, we over-expressed this miRNA in the Raji B-cell line using a synthetic mimic of miR-142-3p and analyzed gene expression 24 hours after the transfection. Four replicates each of miR-142-3p-mimic transfection and apparied control mimic.
Project description:ChIP-seq experiments with the antibody BZ1 directed against the EBV protein BZLF1 prior to and 15 hours post induction with doxycycline (100 ng/ml). The doxycycline regulated conditional BZLF1 allele is encoded by an inducible plasmid stably introduced into Raji cells. Experiments were performed as duplicates.