Project description:Transcriptional profiling of insulin exposed H4IIE cells at different time points (6h-24h) to 100 nM insulin, with untreated solvent control cells (blanks). The central role of hepatic insulin resistance in pandemic metabolic diseases urges investigation of the underlying mechanisms of its development and pathogenesis. Besides genetic susceptibility and lifestyle factors, environmental pollutants are suggested risk factors. In order to appraise the role of the thousands of pollutants we are daily exposed to, stable, robust and high throughput cell-based systems should be developed. In this context, it is of paramount importance to select an in vitro system which mimics in vivo insulin responses as close as possible. Therefore, this study was designed to evaluate if the rat H4IIE hepatoma cell line is a physiologically relevant model to study hepatic insulin responses. DNA microarray analysis, real-time PCR and flow cytometric cell cycle analysis were used to assess the relevance of the insulin response in H4IIE cells. Insulin dose dependently stimulated H4IIE growth. Time dependency of the insulin response was shown with real-time PCR for the known insulin responsive genes: Fasn, Pck1 and Irs2. Based on these results, microarray analysis was performed on H4IIE cells exposed to insulin (100 nM) for 6h and 24h. Genes related to carbohydrate (glycolysis and gluconeogenesis) and lipid metabolism (glycerolipid metabolism, cholesterol synthesis and fatty acid oxidation) were most profoundly afflicted, in accordance with in vivo insulin action in liver. Since changes in carbohydrate and lipid metabolism are pivotal in the pathogenesis of insulin resistance, the presence of a physiological relevant insulin response in H4IIE cells pleads for further testing of its potential use in research on pollutant-driven insulin resistance. Microarray analysis was performed on H4IIE cells exposed to insulin (100 nM) for 6h and 24h. A separate v+2 A-optimal hybridization design was used for each time-point. Using this design each exposure condition is represented by three biological replicates, with each biological replicate analysed in technical duplicate while applying the dye swap principle to correct for dye bias.

Project description:The beneficial effects of glucocorticoids (GCs) in acute lymphoblastic leukemia (ALL) are based on their ability to induce apoptosis. Omics technologies such as DNA microarray analysis are widely used to study the changes in gene expression and have been successfully implemented in biomarker identification. In addition, time series studies of gene expression enable the identification of correlations between kinetic profiles of glucocorticoid receptor (GR) target genes and diverse modes of transcriptional regulation. This study presents a genome-wide microarray analysis of both our and published Affymetrix HG-U133 Plus 2.0 data in GCs-sensitive and -resistant ALL. GCs-sensitive CCRF-CEM-C7-14 cells were treated with dexamethasone at three time points (0 h, 2 h and 10 h). The treated samples were then compared to the control (0 h). The published data used were as follows: GSE2677: GSM51674: B-ALL-24-24h GSM51675: B-ALL-24-6h GSM51676: B-ALL-24-0h GSM51680: B-ALL-17-24h GSM51681: B-ALL-17-8h GSM51682: B-ALL-17-0h GSM51677: B-ALL-13-24h GSM51678: B-ALL-13-8h GSM51679: B-ALL-13-0h GSM51683: B-ALL-31-24h GSM51684: B-ALL-31-6h GSM51685: B-ALL-31-0h GSM51686: B-ALL-32-24h GSM51687: B-ALL-32-6h GSM51688: B-ALL-32-0h GSM51689: B-ALL-33-24h GSM51690: B-ALL-33-6h GSM51691: B-ALL-33-0h GSM51692: B-ALL-37-24h GSM51693: B-ALL-37-6h GSM51694: B-ALL-37-0h GSM51695: B-ALL-38-24h GSM51696: B-ALL-38-6h GSM51697: B-ALL-38-0h GSM51698: B-ALL-40-24h GSM51699: B-ALL-40-6h GSM51700: B-ALL-40-0h GSM51701: B-ALL-43-24h GSM51702: B-ALL-43-6h GSM51703: B-ALL-43-0h GSM51707: T-ALL-25-24h GSM51708: T-ALL-25-6h GSM51709: T-ALL-25-0h GSM51704: T-ALL-20-24h GSM51705: T-ALL-20-8h GSM51706: T-ALL-20-0h GSM51710: T-ALL-2-24h GSM51711: T-ALL-2-8h GSM51712: T-ALL-2-0h GSE2842 GSM60545: S-Line-PreB-6h-EtOH GSM60546: S-Line-PreB-6h-GC GSM60547: S-Line-PreB-24h-GC GSM60542: S-Line-C7H2-6h-EtOH GSM60543: S-Line-C7H2-6h-GC GSM60544: S-Line-C7H2-24h-GC GSM60560: R-Line-CEMC1-6h-EtOH GSM60561: R-Line-CEMC1-6h-GC GSM60562: R-Line-CEMC1-24h-GC GSM60564: R-Line-C7R1-6h-EtOH GSM60566: R-Line-C7R1-6h-GC GSM60576: R-Line-C7R1dim-low-6h-EtOH GSM60578: R-Line-C7R1dim-low-6h-GC GSM60579: R-Line-C7R1dim-low-24h-GC GSM60581: R-Line-PreB-6h-EtOH GSM60583: R-Line-PreB-6h-GC GSM60584: R-Line-PreB-24h-EtOH GSM60586: R-Line-PreB-24h-GC GSM60548: C-Line-CEMC1-ratGR-6h-EtOH GSM60549: C-Line-CEMC1-ratGR-6h-GC GSM60550: C-Line-CEMC1-ratGR-24h-GC GSM60551: C-Line-C7R1dim-high-6h-EtOH GSM60552: C-Line-C7R1dim-high-6h-GC GSM60553: C-Line-C7R1dim-high-24h-GC

Project description:The fungal pathogen Sclerotinia sclerotiorum infects a broad range of dicotyledonous plant species and is the causative agent of stem rot in Brassica napus. To elucidate the mechanisms underlying the defense response, we studied the patterns of gene expression in a partially resistant variety of ZhongYou 821 (ZY821) and a susceptible line from Westar over five time points, 6, 12, 24, 48, and 72 hours post-inoculation (hpi) using a B. napus oligonucleotide microarray. Maximum differential gene expression was observed at 48 hpi in both genotypes with ZY821 responding more quickly than Westar. Specific sets of genes exhibited higher levels of expression at the earlier stages of the infection (6-12 hpi), including genes encoding defense-associated proteins such as chitinases, glucanases, osmotins and lectins and genes encoding transcription factors belonging to the zinc finger, WRKY, AP2, and MYB classes. Genes encoding enzymes involved in JA, ethylene, and auxin synthesis were induced in both genotypes, as were those for gibberellin degradation. Changes in metabolic pathways affecting carbohydrate and energy metabolism appeared to be directed toward shuttling carbon reserves to the TCA cycle. Genes involved in glucosinolate and phenylpropanoid biosynthesis were highly up-regulated suggesting that secondary metabolites are also important components of the response to S. sclerotiorum in B. napus. Keywords: Time course, infected vs mock infected Time course experiment (6hr, 12 hr, 24 hr, 48 hr, 72 hrs), sclerotinia infected vs mock-infected. Biological replicates: 3, independently harvested. Technical replicates: 2, dye swapped within each time for each biological replicate (total 6 slides per time point) time after innoculation: 6 hours: zy 6h inf vs control rep1, zy 6h control vs inf rep1, zy 6h inf vs control rep2, zy 6h control vs inf rep2, zy 6h inf vs control rep3, zy 6h control vs inf rep3 time after innoculation: 12 hours: zy 12h inf vs control rep1, zy 12h control vs inf rep1, zy 12h inf vs control rep2, zy 12h control vs inf rep2, zy 12h inf vs control rep3, zy 12h control vs inf rep3 time after innoculation: 24 hours: zy 24h inf vs control rep1, zy 24h control vs inf rep1, zy 24h inf vs control rep2, zy 24h control vs inf rep2, zy 24h inf vs control rep3, zy 24h control vs inf rep3 time after innoculation: 48 hours: zy 48h inf vs control rep1, zy 48h control vs inf rep1, zy 48h inf vs control rep2, zy 48h control vs inf rep2, zy 48h inf vs control rep3, zy 48h control vs inf rep3 time after innoculation: 72 hours: zy 72h inf vs control rep1, zy 72h control vs inf rep1, zy 72h inf vs control rep2, zy 72h control vs inf rep2, zy 72h inf vs control rep3, zy 72h control vs inf rep3 biological replicate: zy 6h inf vs control rep1, zy 6h inf vs control rep2, zy 6h inf vs control rep3 biological replicate: zy 6h control vs inf rep1, zy 6h control vs inf rep2, zy 6h control vs inf rep3 technical replicate - labeled-extract: zy 6h inf vs control rep1, zy 6h control vs inf rep1 technical replicate - labeled-extract: zy 6h inf vs control rep2, zy 6h control vs inf rep2 technical replicate - labeled-extract: zy 6h inf vs control rep3, zy 6h control vs inf rep3 biological replicate: zy 12h inf vs control rep1, zy 12h inf vs control rep2, zy 12h inf vs control rep3 biological replicate: zy 12h control vs inf rep1, zy 12h control vs inf rep2, zy 12h control vs inf rep3 technical replicate - labeled-extract: zy 12h inf vs control rep1, zy 12h control vs inf rep1 technical replicate - labeled-extract: zy 12h inf vs control rep2, zy 12h control vs inf rep2 technical replicate - labeled-extract: zy 12h inf vs control rep3, zy 12h control vs inf rep3 biological replicate: zy 24h inf vs control rep1, zy 24h inf vs control rep2, zy 24h inf vs control rep3 biological replicate: zy 24h control vs inf rep1, zy 24h control vs inf rep2, zy 24h control vs inf rep3 technical replicate - labeled-extract: zy 24h inf vs control rep1, zy 24h control vs inf rep1 technical replicate - labeled-extract: zy 24h inf vs control rep2, zy 24h control vs inf rep2 technical replicate - labeled-extract: zy 24h inf vs control rep3, zy 24h control vs inf rep3 biological replicate: zy 48h inf vs control rep1, zy 48h inf vs control rep2, zy 48h inf vs control rep3 biological replicate: zy 48h control vs inf rep1, zy 48h control vs inf rep2, zy 48h control vs inf rep3 technical replicate - labeled-extract: zy 48h inf vs control rep1, zy 48h control vs inf rep1 technical replicate - labeled-extract: zy 48h inf vs control rep2, zy 48h control vs inf rep2 technical replicate - labeled-extract: zy 48h inf vs control rep3, zy 48h control vs inf rep3 biological replicate: zy 72h inf vs control rep1, zy 72h inf vs control rep2, zy 72h inf vs control rep3 biological replicate: zy 72h control vs inf rep1, zy 72h control vs inf rep2, zy 72h control vs inf rep3 technical replicate - labeled-extract: zy 72h inf vs control rep1, zy 72h control vs inf rep1 technical replicate - labeled-extract: zy 72h inf vs control rep2, zy 72h control vs inf rep2 technical replicate - labeled-extract: zy 72h inf vs control rep3, zy 72h control vs inf rep3

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Mechanistic evaluation of the insulin response in H4IIE hepatoma cells: New endpoints for toxicity testing?

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