Effects of ionomycin (final concentration of 2 µM) and BAPTA/AM (final concentration of 6 µM) on the RO82 W-1 human thyroid cell line
ABSTRACT: The goal of this study was to identifiy cellular pathways modified by calcium storing in cells presenting a glycolytic metabolism and a poor mitochondrial biogenesis. Keywords: calcium stress response in RO82 W-1 cells In duplicate, cells treated with ionomycin or BAPTA/AM during 96h compared to equivalent DMSO treated cells
Project description:The goal of this study was to identifiy cellular pathways modified by calcium storing in cells presenting a glycolytic metabolism and a poor mitochondrial biogenesis. Keywords: calcium stress response in RO82 W-1 cells Overall design: In duplicate, cells treated with ionomycin or BAPTA/AM during 96h compared to equivalent DMSO treated cells
Project description:Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eIF2. However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a ~4.5-fold general translational repression, the protein coding ORFs of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated uORF that repress translation of the main coding ORF under normal conditions. Site specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely in SLC35A4 and MIEF1) encode functional protein products. Ribosome profiling of sodium arsenite treated cells for examination of translational response to induction of eIF2 phosphoylation
Project description:Recent clinical data suggest that the efficacy of statin treatment in patients with heart failure varies depending on the drugs administered. Therefore, the present study was undertaken to compare murine cardiac gene expression following treatment with four different statins. Statins directly regulated cardiac gene expression in a drug-specific manner in mice. The influence of each statin on the cardiac mRNA expression of various genes, including several genes that might be involved in the development of cardiac disease, was not a general effect of this class of drugs. Male C57BL/6J mice (n = 20) were treated with or without a statin (pitavastatin calcium, pravastatin sodium, rosuvastatin calcium, or atorvastatin calcium hydrate) (n = 4 in each group) for four weeks. Total RNA was isolated from the left ventricle of each animal. The fragmented and labeled amplified cDNA was hybridized to a GeneChip Mouse Genome 430 2.0 Array (Affymetrix). Data and statistical analysis were performed with GeneSpring GX version 11.0 software (Agilent Technologies).
Project description:To further elucidate the mechanism of calcium mediated chemoprevention of colorectal cancer,we have employed whole genome microarray expression profiling as a discovery platform to identify genes that differentially expressed in mice from high calcium group compared those from DMH group.The mice of DMH group were received normal diet (calcium concentration,1.24%) and subcutaneous injection of 1,2-Dimethylhydrazine at a dose of 20 mg/kg once weekly for 20 weeks,the mice of high calcium group were received high calcium diet(calcium concentration,3%) and subcutaneous injection of 1,2-Dimethylhydrazine at a dose of 20 mg/kg once weekly for 20 weeks.The mice were sarcrificed at 24 week.Nine normal colonic mucosa (3 from DMH group, 3 from high calcium group mice with tumors, and 3 from high calcium group mice without tumors) were used for the microarray analysis. The differentially expressed genes were indentified between high calcium group and DMH group,as long as differentially expressed pathways and gene ontology terms.
Project description:In this study we aim to determine the role of IL-4/STAT6 in gene expression in human keratinocytes using RNA-sequencing approach. Human keratinocytes were cultured for 2 or 5 days with calcium chloride to induce terminal differentiation as determined by the expression of epidermal differentiation complex genes. The cells were then stimulated with IL-4 for 3 and 24 hours, or along the 5 days culture period. We observed that IL-4 inhibits fully differentiation of keratinocytes, induces genes involved with production of inflammatory mediators, and reduces the healing capacity of human keratinocytes. Moreover, STAT6 controlled important genes involved with calcium binding, inflammation and epidermis development. Human keratinocytes were differentiated with calcium chloride for 2 days and incubated with media alone or 20ng/ml of recombinant human IL-4 for 3 and 24 hours. Human keratinocytes were differentiated with calcium chloride for 5 days with or wihout recombinant human IL-4 (20ng/ml). Keratinocytes transfected with control or STAT6 siRNA were differentiated with calcium chloride for 2 days and then stimulated with recombinant huma IL-4 for 24 hours.
Project description:Regulatory T (Treg) cells play an indispensable role in immune homeostasis. The development and function of Tregs are dependent on transcriptional factor Foxp3, but how constant expression of Foxp3 is maintained in Tregs is not clear. Here we show that ablation of the conserved non-coding DNA sequence 2 (CNS2) at the Foxp3 locus in mice led to spontaneous lymphoproliferative disease and exacerbation of experimental autoimmune encephalomyelitis (EAE). CNS2 is required for activated Treg cells to maintain elevated Foxp3 expression, which is critical for their suppressor function and lineage stability. Mechanistically, upon TCR stimulation, NFAT binds to both CNS2 and Foxp3 promoter and mediates the interaction between CNS2 and Foxp3 promoter. Our findings demonstrated an essential role for CNS2 in maintaining the stability and function of activated Treg cells and identified NFAT as a key mediator of its function. Gene expression was profiled in T regulatory cells (Treg) in WT and CNS2 knockout mice. CNS2 knockout mice lack a conserved non-coding DNA sequence 2 (CNS2) at the Foxp3 locus. Treg cells were further sorted into Foxp3-high and Foxp3-low populations based on the expression level of Foxp3. mRNA was profiled using RNA-Seq (unstranded, polyA+, SE100) in replicate for each condition
Project description:The effect of dietary calcium and dairy proteins on adipose tissue gene expression profile in diet induced obesity Experiment Overall Design: 9-week-old C57Bl/6J-mice were divided into two groups (n=10/group). The control diet was a standard high-fat diet (60% of energy) low in calcium (0.4%). The whey protein diet was a high-calcium (1.8%) high-fat diet with whey protein isolate. After the 21-week treatment, the adipose tissue transcript profiling (2 mice/group) was carried out using Affymetrix Mouse Genome 430 2.0 array.
Project description:Calcium is an essential macronutrient and plant requires it in large amounts for normal growth and development. This ion participated in innumerable processes and affects nearly all aspects of plant growth and development such as signal transduction, metabolism of lipids, proteins, and carbohydrates, cell growth, cell wall and membrane stabilization. We used whole genome microarrays to determine the transcriptomic profile of rice seedlings exposed to short-term and long term Ca2+ deficiency followed by Ca2+ resupply. Calcium treated seedlings were used for for RNA extraction and hybridization on Agilent microarray platform. Three biological replicates of each sample were used for microarray analysis. We wanted to know the altered expression patterns of calcium-responsive genes majorly involved in metabolic processes, signal transdction pathways, transcriptional regulation, and transport of multiple molecules including Ca2+. Seeds of indica rice were surface sterilized and grown hydroponically in rice growing medium. Plants were grown in the rice growth room under the condition of 16 h light/8 h dark (28°C) photoperiod with 70 % humidity. After 5 days of normal growth, some of the seedlings were transferred to nutrient solution lacking CaCl2 (-Ca2+). After 5 and 14 days, calcium (0.798 mM CaCl2) was resupplied to the plants grown in -Ca2+ medium for 6 h.
Project description:To determine if fibroblasts could be reprogrammed to a keratinocyte phenotype p63+KLF4 or LacZ expressing retroviruses were transduced into primary human neonatal fibroblasts. Global gene expression profiling using U133 plus 2.0 arrays were used to deteremine the extent of reprogramming to a keratinocyte phenoypte upon transduction with p63+KLF4. Fibroblasts transduced with p63+KLF4 were also treated +/- high calcium to determine if treatment with calcium could induce differentiation of these cells. Microarray analysis was also performed on cells treated +/- calcium. For gene expression profiling, cultured human fibroblasts were infected with LacZ or p63+KLF4 expressing retroviruses. p63+KLF4 cells were also treated +/- calcium. Microarray analysis using Affymetrix HG-U133 2.0 plus arrays was performed on duplicate samples.
Project description:The protease activity of the paracaspase MALT1 plays an important role in antigen receptor-mediated lymphocyte activation by controlling the activity of the transcription factor NF-kB and is thus essential for the expression of inflammatory target genes. MALT1 is not only present in cells of the hematopoietic lineage, but is ubiquitously expressed. Here we report that Zymosan or S. aureus stimulation induced MALT1 protease activity in human primary keratinocytes. Human primary keratinocytes were treated for 8 h with solvent control (DMSO), PMA/Ionomycin (P/I) or P/I with MALT1-inhibitor LVSR-fmk. Three biological replicates of each stimualtion were analyzed for gene expression profiles.