Expression profiles of high differentiation-inducible murine erhthroleukemia cells
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ABSTRACT: Murine erythroleukemia (MEL) cells are differentiated by dimethyl sulfoxide (DMSO), hexamethylene bisacetamide (HMBA), or trichostatin A (TSA) treatment. We analyzed expression profiles of high differentiation-inducible (HD) MEL cells during chemical induced differentiation. HD MEL cells were cultured at 1, 6, 12, 24, or 36 hours with 1.5% DMSO, 5.0 mM HMBA, or 30 nM TSA. The RNA of HD-MEL cells before and after chemial treatment were compared. The expression ratio (After chemical treatment/Before chemical treatment) was calculated by average of technical quadruplicate microarray experiments includes two dye-swaps.
Project description:Murine erythroleukemia (MEL) cells are differentiated by dimethyl sulfoxide (DMSOM-oM-<M-^I, hexamethylene bisacetamide (HMBAM-oM-<M-^I or trichostatin A (TSAM-oM-<M-^I treatment. We selsected high differentiation-inducible (HD) and low differentiation-inducible (LD)-MEL cells by recloning of original MEL cells. We screened erythroid differentiation related-genes to compare transcriptome of HD and LD-MEL cells. HD-MEL cells and LD-MEL cells were cultured 6, 12, 24, or 36 hours with 1.0% DMSO, 3.0 mM HMBA, or 15 nM TSA. The RNA of HD-MEL cells and LD-MEL cells were compare same time points of each drug treatment. The expression ratio (HD/LD) was calculated by average of technical quadruplicate microarray experiments includes two dye-swaps.
Project description:Efficient processing of target antigens by the ubiquitin-proteasome-system (UPS) is essential for treatment of cancers by T cell therapies. However, immune escape due to impaired expression of IFN-γ-inducible components of the antigen presentation machinery and consequent inefficient processing of HLA-dependent tumor epitopes can be one important reason for failure of such therapies. Here, we show that repeated short-term co-cultures of Melan-A/MART-1 tumor antigen-expressing melanoma cells with Melan-A/MART-1 (26-35)-specific CTL led to the generation of clones resistant to CTL-mediated cell death. To determine which of the UPS components and its associated pathways was responsible for CTL escape; three UKRV-Mel-15a clones were subjected to microarray gene expression analysis. Three UKRV-Mel-15a-derived melanoma clones were isolated following three repeated short-term exposures to Melan-A/MART (26-35) CTL and harvested for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Murine erythroleukemia (MEL) cells are differentiated by dimethyl sulfoxide (DMSO), hexamethylene bisacetamide (HMBA) or trichostatin A (TSA) treatment. We selsected high differentiation-inducible (HD) and low differentiation-inducible (LD)-MEL cells by recloning of original MEL cells. We screened erythroid differentiation related-genes to compare transcriptome of HD and LD-MEL cells.
Project description:How transcription factors (TFs) cooperate within large protein complexes to allow rapid modulation of gene expression during development is still largely unknown. Here we show that the key haematopoietic LIM-domain-binding protein-1 (LDB1) TF complex contains several activator and repressor components that together maintain an erythroid-specific gene expression programme primed for rapid activation until differentiation is induced. A combination of proteomics, functional genomics and in vivo studies presented here identifies known and novel co-repressors, most notably the ETO2 and IRF2BP2 proteins, involved in maintaining this primed state. The ETO2â??IRF2BP2 axis, interacting with the NCOR1/SMRT co-repressor complex, suppresses the expression of the vast majority of archetypical erythroid genes and pathways until its decommissioning at the onset of terminal erythroid differentiation. Our experiments demonstrate that multimeric regulatory complexes feature a dynamic interplay between activating and repressing components that determines lineage-specific gene expression and cellular differentiation. ChIP-Sequencing profiles of the IRF2BP2, GFI1B and LSD1 proteins were generated using mouse erythroleukemia (MEL) cells. RNA-seq experiments of Irf2bp2-WT, Irf2bp2-KD, Eto2-WT, Eto2-KD, Gfi1b-WT, Gfi1b-KD, Lsd1-WT, Lsd1-KD, MEL-non-induced, and MEL-induced stages were performed using standard RNA-seq protocol. Illumina HiSeq 2000 (standard TruSeq RNA sequencing protocol) was used for the sequencing.
Project description:RNA-seq data were generated for two conditions: for parental SK-MEL-239 cells grown in normal media and resistant SK-MEL-239 cells grown in media supplemented with vemurafenib.
Project description:ChIP-seq analyses were performed in MEL cells expressing BirA alone or BirA and FLAG-Biotin tagged BCL11A (XL isoform). BCL11A chromatin occupancy in MEL cell line.
Project description:We compared the transcriptomes of differentiating cultures of ES cell derived erythroid progentor cells (ES-EP) and murine erythroleukemia (MEL) cells stably transfected with GATA-1 fused to ER. RNA was isolated from duplicate differntiating cultures of MEL and ES-EP using Affymetrix GeneChip Mouse Gene 1.0 ST.
Project description:Gene expression analysis of MEL-18-silenced MCF7 cell lines. MEL-18 is a component of the polycomb repressive complex (PRC)-1, which is a critical epigenetic modulator of stem cell regulation and normal and cancerous development. Accumulating studies have suggested that MEL-18 might act as a tumor suppressor in several human tumors, including breast cancer. Results provide insight into the functional role of MEL-18 in estrogen-dependent breast cancer. MCF7 cells stably infected with lentiviruses encoding either control (shCon) or MEL-18 shRNA (shMEL) were cultured in phenol-red free DMEM supplemented with 10% FBS for 48 h. Total RNA was isolated from the cultures using Trizol reagent. For each of the 2 conditions, 2 biological replicates were included. In total, 4 microarray samples were analyzed; 2 controls and 2 shRNA MEL-18 knockdowns. All labeling, hybridization and scanning steps were performed according to the manufacturers’ instructions.
Project description:We analyzed the transcriptional response of the human melanoma cell line Ma-Mel-15 either transfected with control siRNA (siNT = non-targeting siRNA) or transfected with siRNAs (pool of 4 active and independent siRNAs) directed against the melanocytic transcription factor and lineage oncogene MITF (Microphthalmia-associated transcription factor). The experiment was performed as biological duplicates and RNA was isolated 48 hours after siRNA transfection. We aimed to determine novel markers and pathways of melanoma cell plasticity. Total RNA was obtained from siRNA-treated Ma-Mel-15 melanoma cell lines and global gene expression profiling was done using the Illumina Human HT12 v4 platform.
Project description:Ma-Mel-15 human melanoma cell cultures were transiently transfected (RNAiMax, Lipofectamin) with control siRNA, siRNA against MITF (pool of 4 siRNAs), siRNA against c-JUN (pool of 4 siRNAs) or combinations of siMITF and siJUN. Cells were then either treated with TNF-alpha (1000U/ml) for 24 hours or left untreated. The experiment was performed as biological duplicates. We aimed to determine how c-JUN cooperates with acute MITF-loss in human melanoma cells to increase inflammatory responsiveness and cell plasticity. Total RNA was obtained from siRNA/TNF-treated Ma-Mel-15 melanoma cell lines and global gene expression profiling was done using the Illumina Human HT12 v4 platform.