Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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PPARg dependent expression changes during differentiation of 3T3L1 cell into adipocytes

ABSTRACT: We profiled PPARg dependent gene expression changes during differntiation of 3T3L1 cell using PPARg siRNA 3T3-L1 (Pre-adipocyte) cell line was induced to differentiate using standard adipocyte differentiation media (IBMX, Dex and Insulin) 48hrs post-confluency. RNA was harvested at day -2 (confluent fibroblasts), 48hrs post-induction with IBMX, DEX and Insulin (day=0) and for each subsequent day after rosiglitazone treatment. Illumina beadchip microarrays were used to determine expression profiles of genes differentially regulated in cells transfected with either siRNA targeting PPARgamma or a non-targeting control siRNA. 3T3L1 cell were induced to differentiate into adipocytes using IBMX, DEX and Insulin. RNA from cell treated with PPARg-specific siRNA and non-specific siRNA was isolated at different timepoints. Illumina MouseRef-8 v1.1 Bead chips were used for expression profiling

ORGANISM(S): Mus musculus

SUBMITTER: Mohamed Hamza

PROVIDER: E-GEOD-12929 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

De-novo identification of PPARgamma/RXR binding sites and direct targets during adipogenesis.

Hamza Mohamed Sabry MS Pott Sebastian S Vega Vinsensius B VB Thomsen Jane S JS Kandhadayar Gopalan Srinivasan GS Ng Patrick Wei Pern PW Chiu Kuo Ping KP Pettersson Sven S Wei Chia Lin CL Ruan Yijun Y Liu Edison T ET

PloS one 20090320 3

<h4>Background</h4>The pathophysiology of obesity and type 2 diabetes mellitus is associated with abnormalities in endocrine signaling in adipose tissue and one of the key signaling affectors operative in these disorders is the nuclear hormone transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma). PPARgamma has pleiotropic functions affecting a wide range of fundamental biological processes including the regulation of genes that modulate insulin sensitivity, adipocyte ...[more]

PMID: 19300518

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Project description:Differentiation of 3T3-L1 preadipocytes can be induced by a 2-d treatment with a factor "cocktail" (DIM) containing the synthetic glucocorticoid dexamethasone (dex), insulin, the phosphodiesterase inhibitor methylisobutylxanthine (IBMX) and fetal bovine serum (FBS). We temporally uncoupled the activities of the four DIM components and found that treatment with dex for 48 h followed by IBMX treatment for 48 h was sufficient for adipogenesis, whereas treatment with IBMX followed by dex failed to induce significant differentiation. Similar results were obtained with C3H10T1/2 and primary mesenchymal stem cells. The 3T3-L1 adipocytes differentiated by sequential treatment with dex and IBMX displayed insulin sensitivity equivalent to DIM adipocytes, but had lower sensitivity to isoproterenol-stimulated lipolysis and reduced triglyceride content. The nondifferentiating IBMX–then-dex treatment produced transient expression of adipogenic transcriptional regulatory factors C/EBP{beta} and C/EBP{delta}, and little induction of terminal differentiation factors C/EBP{alpha} and PPAR{gamma}. Moreover, the adipogenesis inhibitor preadipocyte factor-1 (Pref-1) was repressed by DIM or by dex-then-IBMX, but not by IBMX-then-dex treatment. We conclude that glucocorticoids drive preadipocytes to a novel intermediate cellular state, the dex-primed preadipocyte, during adipogenesis in cell culture, and that Pref-1 repression may be a cell fate determinant in preadipocytes. We sought to define the genes differentially expressed in response to dexamethasone (dex) in 3T3L1 mouse preadipocytes. We treated 3T3L1 cells with dex or DMSO vehicle for 2 or 36 hrs and then prepared total RNA. We hybridized each sample (Cy5) to a pool of the samples, which served as the reference (Cy3) channel. All linear models for differential gene expression were constructed with the "limma" package (Smyth, 2004) in BioConductor (Gentleman et al., 2004). A linear model for dex was constructed to compare the 4 biological replicates of DMSO vehicle treatment with dex treatment at either 2 or 36hr. Genes were considered differentially expressed if at least one probe had log-odds greater than 50:50 in the linear model.

Project description:The Forkhead family of transcription factors comprises numerous members and is implicated in various cellular functions, including cell growth, apoptosis, migration and differentiation.In this study we identified the Forkhead factor FoxQ1 as increased in expression during TGF-beta1 induced changes in epithelial differentiation, suggesting functional roles of FoxQ1 for epithelial plasticity.The repression of FoxQ1 in mammary epithelial cells led to a change in cell morphology characterized by an increase in cell size, pronounced cell-cell contacts and an increased expression of several junction proteins (e.g. E-cadherin). In addition, FoxQ1 knock-down cells revealed rearrangements in the actin-cytoskeleton and slowed down cell cycle G1-phase progression.Furthermore, repression of FoxQ1 enhanced the migratory capacity of coherent mammary epithelial cells.Gene expression profiling of NM18 cells indicated that FoxQ1 is a relevant downstream mediator of TGF-beta1 induced gene expression changes. This included the differential expression of transcription factors involved in epithelial plasticity, e.g. Ets-1, Zeb1 and Zeb2.In summary, this study has elucidated the functional impact of FoxQ1 on epithelial differentiation Cells treated with 200µM 4sU for 2h, biological replicate 1:2h_co._total1, Cells treated with 200µM 4sU for 2h, biological replicate 2:2h_co._total2, Cells treated with 200µM 4sU for 2h, biological replicate 3 :2h_co._total3, Cells treated with 200µM 4sU for 2h and enriched for 4sU labelled RNA, biological replicate 1: 2h_co._enriched1, Cells treated with 200µM 4sU for 2h and enriched for 4sU labelled RNA, biological replicate 2: 2h_co._enriched2, Cells treated with 200µM 4sU for 2h and enriched for 4sU labelled RNA, biological replicate 3:2h_co._enriched3, Cells treated with 5 ng/ml TGF-b1 and 200µM 4sU for 2h, biological replicate 1:2h_TGFbeta_total1, Cells treated with 5 ng/ml TGF-b1 and 200µM 4sU for 2h, biological replicate 2:2h_TGFbeta_total2, Cells treated with 5 ng/ml TGF-b1 and 200µM 4sU for 2h, biological replicate 3:2h_TGFbeta_total3, Cells treated with 5 ng/ml TGF-b1 and 200µM 4sU for 2h and enriched for 4sU labelled RNA, biological replicate 1:2h_TGFbeta_enriched1, Cells treated with 5 ng/ml TGF-b1 and 200µM 4sU for 2h and enriched for 4sU labelled RNA, biological replicate 2:2h_TGFbeta_enriched2, Cells treated with 5 ng/ml TGF-b1 and 200µM 4sU for 2h and enriched for 4sU labelled RNA, biological replicate 3:2h_TGFbeta_enriched3, FoxQ1 dataset Cells transfected with 25nm scrambled siRNA for 48hrs, biological replicate 1:ns-siRNA 48hrs 1, Cells transfected with 25nm scrambled siRNA for 48hrs, biological replicate 2:ns-siRNA 48hrs 2, Cells transfected with 25nm scrambled siRNA for 48hrs, biological replicate 3:ns-siRNA 48hrs 3, Cells transfected with 25nm scrambled siRNA for 48hrs and treated with TGF-b1 for 40hrs biological replicate 1:ns-siRNA 48hrs TGF-b1 40hrs 1, Cells transfected with 25nm scrambled siRNA for 48hrs and treated with TGF-b1 for 40hrs biological replicate 2:ns-siRNA 48hrs TGF-b1 40hrs 2, Cells transfected with 25nm scrambled siRNA for 48hrs and treated with TGF-b1 for 40hrs biological replicate 3:ns-siRNA 48hrs TGF-b1 40hrs 3, Cells transfected with 25nm FoxQ1 siRNA for 48hrs and treated with TGF-b1 for 40hrs biological replicate 1:FoxQ1 siRNA 48hrs TGF-b1 40hrs 1, Cells transfected with 25nm FoxQ1 siRNA for 48hrs and treated with TGF-b1 for 40hrs biological replicate 2:FoxQ1 siRNA 48hrs TGF-b1 40hrs 2 Cells transfected with 25nm FoxQ1 siRNA for 48hrs and treated with TGF-b1 for 40hrs biological replicate 3:FoxQ1 siRNA 48hrs TGF-b1 40hrs 3

Dataset Information

PPARg dependent expression changes during differentiation of 3T3L1 cell into adipocytes

Publications

De-novo identification of PPARgamma/RXR binding sites and direct targets during adipogenesis.

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