Search for transcripts encoding secreted proteins whose expression are highly induced after phlebotomy.
Identification of erythroferrone as an erythroid regulator of iron metabolism.
Sex, Age, Specimen part, Time
View SamplesUndifferentiated and differentiated Keratinocytes (AG1478 treated) were stained with antibody-RNA conjugates to measure protein-based diffrentiation changes in conjunction with single-cell transcriptomics. The cells were crosslinked and stained according to the RAID procedure to allow intracellular immunostaining. Antibodies used in this experiment are (TGM1, NOTCH1, KLK6, JAG1, phospho-RPS6, phospho-FAK). Overall design: Three 384 wells plates for untreated and Three 384 wells plates for AG1478 treated cells were processed for single cell transcriptomics
Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells.
Specimen part, Treatment, Subject
View SamplesCell fixation, permeabilization and antibody staining of could have adverse effects on the quality of single cell transcriptomics data. To assess the effects of the RAID procedure, which includes such steps, we performed a direct comparison of single cell transcriptomics by CELseq2 using unfixed and RAID-processed cells. Quality measures (gene complexity, gene detection rate, average gene expression) were performed using 40000 samples UMI counts per cell. Overall design: Single cells were sorted in 96, wells plates. Per condition (unfixed or RAID) three sets (A,B,C) of 48 cells were processed with the CELseq2 protocol.
Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells.
Specimen part, Subject
View SamplesUndifferentiated and differentiated Keratinocytes (AG1478 treated) were stained with antibody-RNA conjugates (targeting EGFR and ITGA6) to measure protein-based differentiation changes in conjunction with single-cell transcriptomics. Overall design: Two 384 wells plates for untreated and two 384 wells plates for AG1478 treated cells were processed for single cell transcriptomics.
Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells.
Specimen part, Treatment, Subject
View SamplesThe mesencephalic dopaminergic (mDA) cell system is composed by two major groups of projecting cells in the Substantia Nigra (A9 neurons) and the Ventral Tegmental Area (A10 cells). A9 neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinsons disease (PD). We used cDNA microarrays and nanoCAGE technology coupled with Laser Capture Microdissection (LCM) to characterize the intrinsic physiological properties of A9 DA neurons. Surprisingly, we found that these cells express alpha- and beta- chains of haemoglobin. Here we report that globin-immunoreactivity decorates the majority of A9 DA neurons, a subpopulation of cortical and hippocampal astrocytes as well as mature oligodendrocytes. This pattern of expression was confirmed in different mouse strains, in rat and human. This is the first report showing that haemoglobin is expressed in the Substantia Nigra of human post mortem brain. Our data suggest that the most famed oxygen-carrying globin is not exclusively restricted to the blood, but it may play a role in the normal physiology of the brain as well as in neurodegenerative disorders.
Unexpected expression of alpha- and beta-globin in mesencephalic dopaminergic neurons and glial cells.
Cell line
View SamplesThe study was designed in order to identify genes differentially expressed when glucocorticoid signaling is blocked by a glucocorticoid-receptor antagonist (RU486 mifepristone) in the context of brain inflammation induced by bacterial lipopolysaccharide (LPS). LPS is only able to cause murine brain damage in our experimental conditions upon RU486 pre-treatment. Hence, the study may reveal potential candidate genes to mediate neuroprotection or neurotoxicity. Due to the factorial design of the experiment, RU486 main-effect could be dissociated from the effects resultant of RU486/inflammation interaction. In addition, brain dissection was conducted to verify the effects in the brain side ipsilateral or contralateral to the site of intracerebral LPS infusion.
Genes involved in the balance between neuronal survival and death during inflammation.
Sex, Age, Specimen part
View SamplesThe purpose was to determine AcP- and AcPb-dependent gene responses to IL-1 by virally-reconstituting AcP-deficient mouse embryonic cortical neurons with CD25 (control), full length AcP, AcPb or the combination of both. A control population was transduced with a CD25-expressing virus. Half the samples were stimulated with IL-1-beta for four hours, RNA was analyzed by microarray.
A central nervous system-restricted isoform of the interleukin-1 receptor accessory protein modulates neuronal responses to interleukin-1.
Specimen part
View SamplesObesity has been shown to increase risk for cardiovascular disease and type-2 diabetes. In addition, it has been implicated in aggravation of neurological conditions such as Alzheimer's. In the model organism Drosophila melanogaster, a physiological state mimicking diet-induced obesity can be induced by subjecting fruit flies to a solid medium disproportionately higher in sugar than protein (HSD) or that has been supplemented with a rich source of saturated fat (HFD). These flies can exhibit increased circulating glucose levels, increased triglyceride content, insulin-like peptide resistance, and behavior indicative of neurological decline, such as decreased climbing ability. We subjected Oregon-R-C flies to variants of the HSD, HFD, or normal (control) diet (ND), followed by a total RNA extraction from fly heads of each diet group for the purpose of Poly-A selected RNA-Sequencing. We targeted at least 50 million paired-end, stranded reads of 75 basepairs in size, and analyzed 4 biological replicates per dietary condition. Our objective was to identify the effects of obesogenic diets on transcriptome patterns, how they differed between obesogenic diets, and identify genes that may relate to pathogenesis accompanying an obesity-like state. Functional annotation and enrichment analysis among genes whose expression was significantly affected by the obesogenic diets indicated an overrepresentation of genes associated with immunity, metabolism, and hemocyanin in the HFD group, and CHK, cell cycle activity, and DNA binding and transcription in the HSD group. Heat map representation of genes affected by both diets illustrated a large fraction of differentially expressed genes between the two diet groups. Diets high in sugar and diets high in fat both have notableeffects on the Drosophila transcriptome in head tissue. The impacted genes, and how they may relate to pathogenesis in the Drosophila obesity-like state, warrant further experimental investigation. Our results also indicate differences in the effects of the HFD and HSD on expression profiles in head tissue of Oregon-R-C flies, despite the reportedly similar phenotypic impacts of the diets. Overall design: Flies were reared on one of three diets (high fat, high sugar, or normal). 6 replicates, with twenty flies each, from each diet treatment were collected for a total of 18 samples. The heads of the flies were then obtained, and RNA extracted from each of those samples. 4 of the RNA samples from each diet group (12 samples total) were sequenced.
RNA-Sequencing of <i>Drosophila melanogaster</i> Head Tissue on High-Sugar and High-Fat Diets.
Specimen part, Subject
View SamplesWTX encodes a tumor suppressor, frequently inactivated in Wilms tumor, with both plasma membrane and nuclear localization. WTX has been implicated in beta-catenin turnover, but its effect on nuclear proteins is unknown. We report an interaction between WTX and p53, derived from the unexpected observation of WTX, p53 and E1B 55K colocalization within the characteristic cytoplasmic body of adenovirus-transformed kidney cells. In other cells without adenovirus expression, the C-terminal domain of WTX binds to the DNA binding domain of p53, enhances its binding to CBP, and increases CBP/p300-mediated acetylation of p53 at Lys 382. WTX knockdown accelerates CBP/p300 protein turnover and attenuates this modification of p53. In p53-reconstitution experiments, cell cycle arrest, apoptosis, and p53-target gene expression are suppressed by depletion of WTX. Together, these results suggest that WTX modulates p53 function, in part through regulation of its activator CBP/p300.
The WTX tumor suppressor enhances p53 acetylation by CBP/p300.
Cell line
View SamplesThe small RNA payload of mammalian sperm undergoes dramatic remodeling during development, as several waves of microRNAs and tRNA fragments are shipped to sperm during post-testicular maturation in the epididymis. Here, we take advantage of this developmental process to probe the function of the sperm RNA payload in preimplantation development. We generated zygotes via intracytoplasmic sperm injection (ICSI) using sperm obtained from the proximal (caput) vs. distal (cauda) epididymis, then characterized development of the resulting embryos. Embryos generated using caput sperm significantly overexpress multiple regulatory factors throughout preimplantation development, and subsequently implant inefficiently and fail soon after implantation. Remarkably, microinjection of purified cauda-specific small RNAs into caput-derived embryos not only completely rescued preimplantation molecular defects, but also suppressed the postimplantation embryonic lethality phenotype. These findings reveal an essential role for small RNA remodeling during post-testicular maturation of mammalian sperm, and identify a specific preimplantation gene expression program responsive to sperm-delivered microRNAs. Overall design: Zygotes were generated by ICSI from sperm isolated from the testis, caput epididymis, or cauda epididiymis. Following fertilization by ICSI zygotes were developed to different stages of preimplantation development and were harvested for single-embryo RNA-Seq (Smart-Seq 2 protocol). For RNA injection experiments 3 hours after fertilization by ICSI RNA was injected using an Eppendorf Femtojet injection setup.
Small RNAs Gained during Epididymal Transit of Sperm Are Essential for Embryonic Development in Mice.
Cell line, Subject
View Samples