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Genes downstream of p53 mediating its effect on megakaryopoiesis

ABSTRACT: In order to examine genes whose expression is altered as a result of the knock-down (KD) of p53, we used gene expression microarray analysis based on the CHRF cell line, a human megakaryoblastic cell line, which upon PMA stimulation undergoes polyploidization, and extends proplatelet-like cytoplasmic protrusions combined with apoptosis. The CHRF cell line has been validated in our laboratory as a good model of in vitro megakaryopoiesis by comparison to hematopoietic stem cells undergoing Mk differentiation. As described, we have previously generated stable p53-KD CHRF cell lines by lentiviral delivery of microRNA-adapted-short hairpin sequences targeting p53 as well as a suitable scrambled-control cell line. For the microarray analysis we utilized the 4x44K whole human genome microarrays from Agilent. This analysis compared the p53-KD CHRF cells against the control CHRF cells in the absence of PMA treatment and on days 1, 3, 5 and 7 after PMA treatment (5 time-points). p53-KD and control samples were hybridized against each other (direct comparison). We carried out two biological replicate experiments, i.e., 10 measurements of gene expression for each microarray probe. Probes from which >50% of measurements were missing were discarded and not included in the analysis of differential expression. Differentially expressed genes were identified using the significance analysis of microarrays (SAM) with a false discovery rate (FDR) of less than 5%, as implemented in the MultiExperiment Viewer (MeV) tool.

ORGANISM(S): Homo sapiens

SUBMITTER: Eleftherios Papoutsakis

PROVIDER: E-GEOD-30984 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Genes downstream of p53 mediating its effect on megakaryopoiesis

Project description:In order to examine genes whose expression is altered as a result of the knock-down (KD) of p53, we used gene expression microarray analysis based on the CHRF cell line, a human megakaryoblastic cell line, which upon PMA stimulation undergoes polyploidization, and extends proplatelet-like cytoplasmic protrusions combined with apoptosis. The CHRF cell line has been validated in our laboratory as a good model of in vitro megakaryopoiesis by comparison to hematopoietic stem cells undergoing Mk differentiation. As described, we have previously generated stable p53-KD CHRF cell lines by lentiviral delivery of microRNA-adapted-short hairpin sequences targeting p53 as well as a suitable scrambled-control cell line.

2011-07-27 | GSE30984 | GEO

Transcription profiling of human CHRF-288 cell line after phorbol ester stimulation

Project description:Little is known about the global transcriptional program underlying megakaryocytic (Mk) differentiation, maturation, and apoptosis. Using DNA microarrays and Q-RT-PCR, we examined the transcriptional profile of phorbol-ester-induced Mk differentiation of the megakaryoblastic CHRF-288-11 (CHRF) cell line â a model system for investigating megakaryopoiesis. The goals of this study were to (1) verify the megakaryocytic nature of the CHRF cell line at the transcriptional level, and (2) extract novel insights into the key facets of Mk maturation including polyploidization, proplatelet formation, and apoptosis. Experiment Overall Design: CHRF-288 cells were cultured in the presence of 10 ng/mL phorbol ester (PMA) or equivalent volume of DMSO solvent (0.02%). Unstimulated control cells from time zero (exponentially growing CHRF cells) were also analyzed. For PMA treated legs, only the adherent cells were included in the transcriptional analysis. Two biological replicate experiments were analyzed and approximately one-half of the samples with each experiment were technically replicated. Hybridizations were performed in a reference design with all samples labeled with Cy3 and a reference RNA pool labeled with Cy5.

2008-06-12 | E-GEOD-3838 | biostudies-arrayexpress

Transcription profiling of CHRF-288-11 and primary human megakaryocytic cell cultures provide novel insights into lineage-specific differentiation

Project description:This SuperSeries is composed of the following subset Series: GSE3838: Temporal expression profile of CHRF-288 cell line after phorbol ester stimulation CHRF-288 cells were cultured in the presence of 10 ng/mL phorbol ester (PMA) or equivalent volume of DMSO solvent (0.02%). Unstimulated control cells from time zero (exponentially growing CHRF cells) were also analyzed. For PMA treated legs, only the adherent cells were included in the transcriptional analysis. Two biological replicate experiments were analyzed and approximately one-half of the samples with each experiment were technically replicated. Hybridizations were performed in a reference design with all samples labeled with Cy3 and a reference RNA pool labeled with Cy5. GSE3839: Temporal expression profile of megakaryocytic differentiation primary CD34+ cell culture Experiment G-CSF mobilized peripheral blood CD34-positive cells were cultured with TPO, IL-3, and Flt3-L to induce Mk differentiation. Samples prior to day 5, including uncultured starting cells, were analyzed directly, whereas samples after and including day 5 were positively selected for CD41a expression immediately prior to RNA isolation. Three biological replicate experiments were analyzed and approximately one-half of the samples from each experiment were technically replicated. Hybridizations were performed in a reference design with all samples labeled with Cy3 and a reference RNA pool labeled with Cy5.

2008-01-18 | E-GEOD-8914 | biostudies-arrayexpress

FAIRE-seq of the human megakaryocytic cell line CHRF-288-11

Project description:FAIRE-seq of the human megakaryocytic cell line CHRF-288-11

2012-01-11 | E-MTAB-858 | biostudies-arrayexpress

MEIS1 ChIPseq in CHRF-288-11

Project description:ChIP-seq of MEIS1 in the human megakaryocytic cell line CHRF-288-11

2012-01-04 | E-MTAB-859 | biostudies-arrayexpress

Gene expression analysis of TRIM5 KD in the THP1 cell line

Project description:We had evidence that TRIM5 regulates signal transduction, specifically NFkB and MAPK pathways. To test the role of endogenous TRIM5 we used the myelomonocytic leukemia cell line THP1. These cells were transduced with a lentiviral vector that delivers a miRNA engineered to knockdown TRIM5. The vector also encoded a puromycin-resistance cassette and transduced cells were selected in poold with puromycin. As a control, cells were transduced with a vector targeting luciferase instead of TRIM5. After selection in puromycin, the TRIM5 KD and Luciferase KD cells were differentiated into macrophages by treatment with PMA. 3 days later, RNA was harvested for analysis.

2010-11-02 | E-GEOD-25041 | biostudies-arrayexpress

Differential gene expression in human THP-1 cell line undifferentiated, 3 days or 8 days differentiated with phorbol myristate acetate (PMA)

Project description:We analysed the capacity of THP-1 cells (differentiated to macrophagoid cells) to recognize RNA sequences via pattern recognition receptors in vitro. Gene expression was analysed by RNA-Microarray. Cytokine production was analysed by ELISA assays. We used microarrays to investigate differential gene expression in THP-1 cell line undifferentiated in comparison with 3 days or 8 days differentiated with phorbol myristate acetate (PMA). Microarray analysis revealed differential gene expression patterns of THP-1 when differentiated. THP-1 cells, undifferentiated, 3 days PMA-differentiated and 8 days PMA-differentiated

2015-11-09 | E-GEOD-67264 | biostudies-arrayexpress

Dose-dependent phorbol 12-myristate-13-acetate mediated monocyte-to-macrophage differentiation induces unique proteomic signatures in THP1 cells

Project description:Macrophages are sentinels of the immune system and THP1 monocytic cells are one of the widely used models to study immune responses in macrophages. Several monocyte-to-macrophage differentiation protocols exist with phorbol 12-myristate-13-acetate (PMA) being widely used and accepted. However, the concentrations and durations of PMA treatment to induce differentiation varies widely in published literature. In this study, we determined the proteome expression dynamics of THP1 macrophages differentiated from monocytes by three commonly used PMA-based differentiation protocols using dimethyl labeling-based quantitative proteomics analysis. Our analysis shows that variations in PMA concentration and varying periods of rest post-stimulation result in downstream differences in proteome expression and cellular processes. We demonstrate that these differences result in altered expression of cytokines upon stimulation with different TLR ligands. Together, these findings provide a valuable resource that significantly expands the knowledge of protein expression dynamics in in vitro models of macrophages which in turn has profound impact on the immune responses being studied.

2021-09-08 | PXD015872 | Pride

C16-ceramide is a natural regulatory ligand of p53 in cellular stress response

Project description:Ceramides are important participants of signal transduction, regulating fundamental cellular processes. Here we report the mechanism for activation of p53 tumor suppressor by C16-ceramide. C16-ceramide tightly binds within the p53 DNA binding domain (Kd ~ 60 nM), in close vicinity to the Box V motif. This interaction is highly selective towards the ceramide acyl chain length with its C10 atom being proximal to Ser240 and Ser241. Ceramide binding stabilizes p53 and disrupts its complex with E3 ligase MDM2 leading to the p53 accumulation, nuclear translocation and activation of the downstream targets. This is a novel physiological mechanism of p53 activation, which is fundamentally different from the canonical p53 regulation through protein-protein interactions or post-translational modifications. The discovered mechanism is triggered by serum or folate deprivation implicating it in the cellular response to nutrient/metabolic stress. Our study establishes C16-ceramide as the first natural small molecule activating p53 through the direct binding.

2018-10-16 | PXD010783 | Pride

Human p53 interating protein ID

Project description:Wild type and mutant p53 were transfected in H1299 cells and the associated complexes were affinity purified. The p53 interacting proteins then analyzed by ms/ms.

2019-01-29 | PXD012463 | Pride

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