Transcriptional profiling of lung tissue of rat pretreated with cobalt in normoxic and hypoxic conditions and comapring with saline treated normoxic and hypoxic lung tissue in different time interval
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ABSTRACT: The goal of the study was to examine the effect of hypoxic preconditioning with cobalt on global gene profiling in lung tissues. Agilent two-color experiment,Organism: Rat ,Slides, Agilentâs Whole Rat Genome Microarray 4x44k GPL4135 with 44000 features, Labeling kit: Agilents Low input RNA linear amplification Kit Cat # 5184-3523, Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA Rat lung tissue: cobalt + Hypoxia (0h, 6h, 12h, 24h, 48h) vs. Hypoxia (0h, 6h, 12h, 24, 48h)
Project description:To identify new primary HIF target genes we cultured HeLa cells under short period (6 hours) of mild hypoxia (1% oxygen) Hypoxia-induced gene expression in HeLa cells was measured after 6h exposure to 1% oxygen and compare to normoxic
Project description:Many primary tumours have low levels of molecular oxygen (hypoxia), and hypoxic tumours respond poorly to therapy. Pan-cancer molecular hallmarks of tumour hypoxia remain poorly understood, with limited comprehension of its associations with specific mutational processes, non-coding driver genes and evolutionary features. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumour types, we quantify hypoxia in 1188 tumours spanning 27 cancer types. Elevated hypoxia associates with increased mutational load across cancer types, irrespective of underlying mutational class. The proportion of mutations attributed to several mutational signatures of unknown aetiology directly associates with the level of hypoxia, suggesting underlying mutational processes for these signatures. At the gene level, driver mutations in TP53, MYC and PTEN are enriched in hypoxic tumours, and mutations in PTEN interact with hypoxia to direct tumour evolutionary trajectories. Overall, hypoxia plays a critical role in shaping the genomic and evolutionary landscapes of cancer.
Project description:RAD18 and RAD6 were co-expressed in E.coli, RAD18 harbors an N-terminal 6His tag which was utilized for affinity chromatography via cobalt affinity resin. The complex was then subjected to size exclusion chromatography. 5 X molar excess MAGEA4 (also has N-terminal His-tag) was added to RAD18-RAD6. The complex was crosslinked with 0.5 x Molar BS3 to lysine residues within the complex for 10 minutes at room temperature, shaking at 600 rpm. The reaction was quenched with 100 mM Tris pH 8, incubated at 35 °C for 10 minutes, shaking at 600 rpm.
Project description:Outcome prediction classifiers were successfully constructed through expression profiling of a total of 1,329 miRNAs in MKN1, gastric cancer cell line under normoxic and hypoxic conditions. In the study presented here, MKN1 under normoxic and hypoxic conditions was used to acquire expression profiles of a total 1,329 unique miRNAs.
Project description:The initiation of donor derived dendritic cell (DC) adhesion and migration are one of the key events mediated by ischemia reperfusion injury (IRI) following solid organ transplantation. Recently we have been able to demonstrate that a single donor treatment with the synthetic metalloporphyrin cobalt protoporphyrin (CoPPIX) for heme oxygenase 1 (HO-1) induction results in the reduction of donor-derived DCs following IRI thus leading to the preservation of graft long-term function. Gene expression analysis in murine liver revealed the up-regulation of the signal transducer and activator of transcription 3 (STAT3) after CoPPIX treatment. Since it has been shown that CoPPIX is able to inhibit DC maturation, we could demonstrate that DC (-LPS) induced maturation guided by the secretion of pro-inflammatory cytokines and alloreactive T cell proliferation is dependent of STAT3 phosphorylation and independent of HO-1 activity. These observations highlight the immunomodulatory capacity of STAT3 which might be of further interest for the inhibition of immune response. Keywords: dendritic cells, heme oxygenase-1 (HO-1), STAT3, cobalt protoporphyrin For the analysis of gene expression profiles in vivo C57BL/6 mice were treated either with 5 mg/kg CoPPIX, i.p., 50 mg/kg MC, p.o. and 500 ppm CO for 6 or 24 hrs (3 animals in each group). Untreated C57BL/6 mice were used as controls (n=9). After 6 or 24 hrs of treatment animals were sacrificed and whole livers were harvested for analysis.
Project description:Injuries, high altitude, and endurance exercise lead to hypoxic conditions in skeletal muscle and sometimes to hypoxia-induced local tissue damage. Thus, regenerative myoblasts/satellite cells are exposed to different levels and durations of partial oxygen pressure depending on the spatial distance from the blood vessels. To date, it is unclear how hypoxia affects myoblasts proliferation, differentiation, and particularly fusion with normoxic myoblasts. To study this, we investigated how 21% and 2% oxygen affects C2C12 myoblast morphology, proliferation, and myogenic differentiation and evaluated the fusion of normoxic- or hypoxic-preconditioned C2C12 cells in 21% or 2% oxygen in vitro. Out data show that the long-term hypoxic culture condition does not affect the proliferation of C2C12 cells but leads to rounder cells and reduced myotube formation when compared with myoblasts exposed to normoxia. However, when normoxic- and hypoxic-preconditioned myoblasts were differentiated together, the resultant myotubes were significantly larger than the control myotubes. Whole transcriptome sequencing analysis revealed several novel candidate genes that are differentially regulated during the differentiation under normoxia and hypoxia in mixed culture conditions and may thus be involved in the increase in myotube size. Taken together, oxygen-dependent adaption and interaction of myoblasts may represent a novel approach for the development of innovative therapeutic targets.
Project description:Hypoxic water had 5 mg/L of Dissolved Oxygen (DO) and normoxic water had 11.5 mg/L DO. 40 immature rainbow trout (about 100 g) were housed for 7 days under these conditions (20 fish in each treatment). Then fish were anesthetized with 100 mg/L MS-222, and blood was sampled using caudal puncture with heparinized syringes. The blood was centrifuged at 10 000 x g for 4 minutes in Heraeus Fresco 21 centrifuge (Thermo Fisher). Plasma was removed from this using a pipette, and placed in cryotubes, flash frozen in liquid nitrogen, and then stored at -80oC until preparation for proteomic analysis.
Project description:Exosomes derived from NSCLC cells under normoxic or hypoxic conditions were compared to identify hypoxic associated enrichment of proteins in exosomes.