Human Differentiated SH-SY5Y neuroblastoma cells treated with Granzyme K
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Brain, Generation Of Neurons
DISEASE(S): Brain Cancer
SUBMITTER: 
daniela sorio
LAB HEAD: Gabriela Constantin
PROVIDER: PXD064640 | Pride | 2025-06-24
REPOSITORIES: Pride
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Project description:The Spi1/ Pu.1 transcription factor plays a crucial role in myeloid cell development across many species. Several Spi1 target genes have been identified so far, yet the Spi1-dependent gene group remains largely unknown. To identify novel genes downstream of Spi1 we employed a microarray strategy using zebrafish embryos. We established the gene group down-regulated upon spi1 knockdown while simultaneously enriched in FACS-sorted embryonic myeloid cells of a spi1:GFP transgenic line, thus representing putative myeloid-specific Spi1 target genes. This gene group contained all previously identified Spi1-dependent zebrafish genes, confirming the validity of the approach, as well as novel immune-related genes. Colocalization studies with neutrophil and macrophage markers revealed that genes cxcr3.2, mpeg1, ptpn6 and mfap4 were expressed specifically in early embryonic macrophages. The analysis of adult zebrafish hematopoietic tissue showed that genes mfap4 and mpeg1 remained macrophage specific within the myeloid fraction throughout zebrafish life. We also demonstrated that gene cxcr3.2, coding for chemokine receptor 3.2, functions in macrophage migration to the site of bacterial infection. These results establish a myeloid-specific gene group dependent on Spi1 in zebrafish and identify novel early macrophage-specific marker genes, which will facilitate further studies of macrophage development and innate immune function. Zebrafish strains Zebrafish (Danio rerio) were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (http://ZFIN.org). The spi1-gfp transgenic line used in this study (Pu1-lynEGFP, a gift from Franscesca Peri, EMBL, Heidelberg) contains 4 kb of spi1/pu.1 promoter sequence driving expression of membrane-targeted EGFP. Morpholino knock-down experiments Morpholino oligonucleotides (Gene Tools) were diluted to required concentration in 1x Danieu’s buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES; pH 7.6) containing 1% Phenol red (Sigma) and approximately 1 nl was injected into the 1-2 cell stage embryo using a Femtojet injector (Eppendorf). Embryo dissociation and FACS (Fluorescent activated cell sorting) Dissociation of embryos was performed according to Covassin et al. 30. In short, ~300 embryos of spi1:GFP line at 28 hpf were dechorionated by pronase treatment, rinsed in calcium free Ringer solution, followed by digestion with 0.25% trypsin. The obtained cell suspension was centrifuged, rinsed with PBS and resuspended in Leibovitz medium L15 without phenol red, 1% fetal calf serum, 0.8mM CaCl2, penicillin 50 U/ml and streptomycin 0.05 mg/ml. The single cell suspension was subject to FACS at room temperature using a FACSAria (Becton Dickinson) with the BD FACSDiva software version 5.0.3 and a Coherent Sapphire solid state laser 488 nm with 13 mW power. The GFP+ and GFP- cell fractions were collected separately into L15 medium supplemented with 0.8 mM CaCl2, 10% fetal calf serum and penicillin 50 U/ml and streptomycin 0.05 mg/ml. The standard yield from circa 300 embryos was approximately 2 x 105 GFP+ cells. Microarray experiment design and analysis Embryos injected either with 1 mM Pu1 morpholino19 or 1 mM Standard Control morpholino were harvested at 28 hours post fertilization (hpf). As a control, embryos injected with an equal volume of 1% Phenol red in Danieu’s buffer (1xPR/D) were used. Pools of 20-30 embryos were collected. RNA samples from spi1 morphants and standard control morphants were labelled with Cy5 and hybridized against a Cy3-labelled common reference (a mixture of all samples injected with 1xPR/D). This experiment was performed in triplicate. FACS sorted spi1-GFP zebrafish embryos at 28hpf were analyzed as follows: the RNA of GFP-positive fraction was labelled with Cy5 and hybridized against a Cy3-labelled RNA of corresponding GFP-negative fraction of cells from the same pool of embryos. This experiment was performed in duplicate. RNA isolation, synthesis of amino allyl labeled aRNA, coupling of Cy3 and Cy5 dyes, and hybridization conditions were as described31. Microarray analysis was performed using custom-designed 44k Agilent chips (platform accession number in GEO: GPL7735) described elsewhere 31. Microarray data were processed and analyzed as described 31. Significance cut-offs for differentially expressed probe sequences were set at 1.2 fold change at P <10-2.
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2023-02-27 | PXD040391 | Pride
Project description:To characterise the transcriptional response in brain of Sockeye Salmon (Oncorhynchus nerka) that were persistently infected with infectious hematopoietic necrosis virus (IHNV) and to determine whether carrying the IHNV affects the ability to respond to other immunological challenges we compared the brain transcriptome of IHNV carriers, IHNV-negative survivors, and naïve Sockeye Salmon that were never exposed to IHNV. In addition we determined the transcriptional changes among carriers, survivors and naïve fish in their response to the viral mimic polyriboinosinic polyribocytidylic acid (poly(I:C)), using the cGRASP 44K salmon oligoarray. Fish that survived an immersion challenge with IHNV and time-matched control (naïve) fish that were never exposed to IHNV were reassigned into 2 tanks each at 271 days post challenge (dpc). One group each of the challenged and naïve fish received an intraperitoneal injection of poly(I:C). For controls, a group each of challenged and naïve fish were not injected. Brain tissues were sampled from each tank at 3 (274 dpc), 7 (278 dpc) and 10 (281 dpc) days after poly(I:C) injection. Based on the presence of IHNV in brain fish were assigned into “carriers” and “survivors”, i.e. fish with detectable and undetectable levels of IHNV, respectively. For transcriptomic profiling brain samples were analysed from naïve (n = 7), survivors (n = 7), and carriers (n = 5), as well as poly(I:C)-injected naïve (n = 7), poly(I:C)-injected survivors (n = 7), and poly(I:C)-injected carriers (n = 2). All analysed brain samples were collected 274 dpc (i.e. 3 d after poly(I:C) injection) with the exception of two carriers that were collected 278 dpc. The smaller sample size for carriers was a result of the few individuals that survived the exposure that ended up carrying the virus.
2015-08-10 | E-GEOD-65241 | biostudies-arrayexpress
Project description:Since 2008, the consumption of the plasticizer bis (2-ethylhexyl) phthalate (DEHP) in the manufacture of plastic products has been restricted due to several adverse health effects. 1,2-cyclohexanedicarboxylic acid diisononyl ester (DINCH) is increasingly used as a harmless substituent for DEHP. However, the structural similarity to DEHP and first studies demonstrating adverse effects on reproduction and metabolism in rats, suggest that DINCH may act adverse on human health. Still, there is a lack of studies on the biological effects of DINCH, especially in the immune system, which is in constant communication with other tissues and thus crucial for maintaining a functional organism. Here we aimed to investigate the effects of DINCH and its naturally occurring biotransformation product monoisononylcyclohexane-1,2-dicarboxylic acid ester (MINCH) on the innate immune response. Human THP-1 macrophages were exposed to DINCH and MINCH in a concentration range of 10 nM to 10 μM for 4 h, 16 h and 24 h in the presence of LPS.
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Project description:DNA processing chain A (DprA) is a DNA binding protein which is ubiquitous in bacteria and required for DNA transformation. However, the interaction of DprA with competence proteins and recombination is poorly understood. Therefore, the proteomes of whole Neisseria meningitidis (Nm) wildtype and dprA mutant cells were compared. Such a comparative proteomic analysis increases our understanding of the interactions of DprA with other Nm components, and may elucidate its potential role beyond DNA processing in transformation. Using label-free quantitative proteomics, a total of 1057 unique Nm proteins were identified out of which 100 were quantified as differentially abundant (P ≤ 0.05 and fold change ≥ |2|) in the dprA null mutant. Proteins involved in homologous recombination (RecA, UvrD and HolA), pilus biogenesis (PilG, PilT1, PilT2, PilM, PilO, PilQ, PilF and PilE), and cell division, including core energy metabolism and response to oxidative stress were downregulated in dprA null mutant Nm. Immunoblotting was employed to validate the differences observed in two selected proteins. The analysis revealed reduced amounts of PilG in the dprA null mutant and reduced amounts of DprA in the Nm pilG null mutant. In conclusion, DprA interacts with proteins essential for Nm DNA recombination in transformation, pilus biogenesis and other functions associated with the inner membrane. Inverse downregulation of Nm DprA and PilG expression in the corresponding mutants indicate a link between DNA processing and pilus biogenesis demonstrated for the first time.
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2022-04-19 | PXD028412 | Pride
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