Real-time quantitative PCR analysis of 3T3-L1 cells treated with Interleukin-15 (IL-15)
ABSTRACT: Differentiated 3T3-L1 adipocytes were either treated 0ng/ml or 500ng/ml rmIL-15 for 24 hours (n=3/group) qPCR gene expression profilling performed in vitro using recombinant murine (rm)IL-15 treated (500ng/ml) and untreated (control) 3T3-L1 cells. Equal amounts of total RNA per subject were used in the assay.
Project description:Differentiation of 3T3-L1 cells into adipocytes involves a highly orchestrated series of events including clonal expansion, growth arrest and terminal differentiation. The mechanisms coordinating these different steps are not yet fully understood. Here we investigated whether micro (mi)RNAs play a role in this process. Microarray analysis was performed to detect miRNA expression during 3T3-L1 preadipocyte differentiation. Several miRNAs, including let-7, were up-regulated during 3T3-L1 adipogenesis. Ectopic introduction of let-7 into 3T3-L1 cells inhibited clonal expansion as well as terminal differentiation. The mRNA encoding high mobility group AT-hook 2 (HMGA2), a transcription factor that regulates growth and proliferation in other contexts, was inversely correlated with let-7 levels during 3T3-L1 cell adipogenesis, and let-7 markedly reduced HMGA2 concentrations. Knockdown of HMGA2 inhibited 3T3-L1 differentiation. These results suggest that let-7 plays an important role in adipocyte differentiation and that it does so in part by targeting HMGA2, thereby regulating the transition from clonal expansion to terminal differentiation. 3T3-L1 cells were induced to differentiation into mature adipocytes using a canonical DMI cocktail. The time point at two days after confluency of 3T3-L1 was defined as day 0. Samples were collected at day 0, day 1, day 4, and day 7. The expression of microRNAs at day 1, day 4, and day 7 was compared to that of day 0.
Project description:Using RNA-Seq, we compared the transcriptomes of differentiated 3T3-L1 adipocytes for control and ZFP407-deficient cells Differentiated 3T3-L1 cells were electroporated with control or 1 of 2 Zfp407 siRNAs. Six independent siRNA electroporations were conducted for the control siRNA and 3 independent electroporations were conducted for each Zfp407 siRNA.
Project description:Human pluripotent stem (hPS) cells (both embryonic and induced) have emerged as a powerful tool for developmental biology studies, genetic and chemical screens, and regenerative medicine for neurological diseases, as they can generate different neuronal subtypes. We performed genome-wide expression profiling in the embryonic stem cells in three developmental stages (day0, day 15 and day 45).
Project description:This SuperSeries is composed of the following subset Series: GSE18598: Differentiating 3T3-L1 adipocytes, introduced with siRNA against aof2 and rfk genes, or treated with tranylcypromine GSE18599: Differentiating 3T3-L1 adipocytes, introduced with siRNA against phf21a gene Refer to individual Series
Project description:Enhancers are developmentally-controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. Here, we show by genome-wide mapping that the newly discovered DNA modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells as well as during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates like Meis1 in P19 cells and PPARgamma in 3T3-L1 cells. Accordingly, a fraction of hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 5mC hydroxylase Tet1. In addition, kinetic studies of cytosine hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation is an early event of enhancer activation. Hence, acquisition of 5hmC in cell-specific distal regulatory regions may represent a major event of enhancer progression toward an active state and participate in selective activation of tissue-specific genes Genome-wide 5hmC distribution was determined using hMeDIP-seq. Cells used in this study are P19.6 mouse embryonal carnicoma cells and P19.6 cells treated for 48 hours with 1µM all-trans retinoic acid (RA), as well as 3T3-L1 cells and 3T3-L1 derived adipocytes differentiated with dexamethasone, insulin and IBMX (differentiation cocktail - DC). Individual hMeDIP samples from P19.6 ord 3T3-L1 cells were pooled for library preparation. Libraries were prepared and sequenced at the IBL sequencing facility (Lille, France) with an Illumina Genome Analyser II.
Project description:Lipid mobilization (lipolysis) in white adipose tissue (WAT) critically controls lipid turnover and adiposity in humans. While the acute regulation of lipolysis has been studied in detail, the transcriptional determinants of WAT lipolytic activity remain still largely unexplored. Here we show that the genetic inactivation of transcriptional co-factor transducin beta-like-related (TBLR) 1 blunts the lipolytic response of white adipocytes through the impairment of cAMP-dependent signal transduction. Indeed, mice lacking TBLR1 in adipocytes are defective in fasting-induced lipid mobilization and when placed on a high fat diet show aggravated adiposity, glucose intolerance and insulin resistance. TBLR1 levels are found to increase under lipolytic conditions in WAT of both human patients and mice, correlating with serum free fatty acids (FFA). As a critical regulator of WAT cAMP signaling and lipid mobilization, proper activity of TBLR1 in adipocytes may thus represent a critical molecular checkpoint for the prevention of metabolic dysfunction in subjects with obesity-related disorders. We used microarrays to identify global gene expression in 3T3-L1 adipocytes lacking TBLR1 and compared gene expression to control shRNA treated cells in both basal and isoproterenol stimulated states. We analyzed 12 RNA samples extracted from 3T3-L1 adipocytes that were treated with either control or TBLR1 specific shRNAs and with or without 10 µM isoproterenol for 3 hrs. Three replicates of each condition.
Project description:Growing evidence indicates that PPARγ agonists, such as rosiglitazone (RSG,), induce adipose mitochondrial biogenesis. Using microarrays, we systematically analyzed nucleus-encoded mitochondrial gene expression in two common murine adipocyte models, 3T3 L1 and C3H/10T1/2 adipocytes, and aimed to further establish the direct role of RSG, and capture the temporal changes in mitochondrial gene transcription during this process. Experiment Overall Design: Fully differentiated 3T3 L1 and C3H/10T1/2 adipocytes were treated with RSG, or DMSO vehicle for 1, 2, 4, 7, 24, and 48 hrs, and total RNA was extracted for microarray analysis.
Project description:PPARγ is a master transcriptional regulator of adipogenesis. Hence, the identification of PPARγ coactivators should help reveal mechanisms controlling gene expression in adipose tissue development and physiology. We show that the non-coding RNA Steroid receptor RNA Activator, SRA, associates with PPARγ and coactivates PPARγ-dependent reporter gene expression. Overexpression of SRA in ST2 adipocyte precursor cells promotes their differentiation into adipocytes. Conversely, knockdown of endogenous SRA inhibits 3T3-L1 preadipocyte differentiation. Microarray analysis reveals hundreds of SRA-responsive genes in adipocytes, including genes in cell cycle, insulin and TNFα signaling pathways. Some functions of SRA may involve mechanisms other than coactivation of PPARγ. SRA increases insulin-stimulated glucose uptake in adipocytes. SRA promotes S-phase entry during mitotic clonal expansion, decreases expression of cyclin-dependent kinase inhibiters p21Cip1 and p27Kip1, and increases phosphorylation of Cdk1/Cdc2. SRA also inhibits the TNFα-induced phosphorylation of c-Jun NH2-terminal kinase. In conclusion, SRA enhances adipogenesis and adipocyte function through multiple pathways. Total RNA was isolated from fully differentiated (MDIT day 4) SRA overexpressing (pMSCV-SRA) and control (pMSCV empty vector) ST2 adipocytes, or fully differentiated (MDIT day 8) shSRA knockdown (pSuperior-shSRA) or shControl (pSuperior-shcontrol) 3T3-L1 adipocytes. Genome wide gene expression analysis was performed using Affymetrix mouse genome 430 2.0 arrays. Triplicate samples were analyzed.