Overexpression of spliced XBP1 in cultured Adipocytes
ABSTRACT: We overexpressed the spliced form of transcription factor XBP1 in mature F442A adipocytes by adenoviral infection. Control virus expressed GFP alone. mouse sXBP1 overexpressed in mouse F442A adipocytes compared to control (GFP alone). Four replicates of each treatment were analyzed.
Project description:Genome-wide transcriptome analysis of genetic pertubations induced in Drosophila notum by the over-expression of the transcription factors Achaete, Asense, E(spl)m3-HLH and Senseless. Transient over-expression of GFP (control), Achaete (Ac), Senseless (Sens), Asense (Ase) and E(spl)m3-HLH transcription factors in Drosophila notum using sca-Gal4, tub-Gal80ts. Transcription factorshave been over-expressed alone or in combinations. Three biological replicates were performed for each conditions.
Project description:Comparing gene expression profile in 3T3-F442A adipocytes with shRNA against TRPV4 or GFP. TRPV4 is an ion channel expressed in adipocytes. Results provided information that how loss-of-function of TRPV4 affects gene expression in adipocytes. 4 samples were analyzed as two groups: shGFP (control) and shTRPV4 (experimental). Each group has two replicates.
Project description:A comparison of the mRNAs analysis in lactation mammary myoepithelial cells (GFP+/GFP-) and brown adipocytes (GFP+) from Myf5-Cre-ROSAmTmG and Ucp1-iCre-ROSAmTmG mice. Results provide the gene expression signature in the brown origin (UCP1/Myf5-positive) myoepithelial cells in vivo. Overall design: Mammary myoepithelial cells and brown adipocytes were isolated by FACS sorting based on the cell surface markers and total RNA were immediately. GFP+ mammary myoepithelial cells (Lin−:CD24+:CD29hi) from lactation day 10 Myf5-Cre-ROSAmTmG mice (n=2). GFP- mammary myoepithelial cells (Lin−:CD24+:CD29hi) from lactation day 10 Myf5-Cre-ROSAmTmG mice (n=2). GFP+ mammary myoepithelial cells (Lin−:CD24+:CD29hi) from lactation day 10 Ucp1-iCre-ROSAmTmG mice (n=2). GFP- mammary myoepithelial cells (Lin−:CD24+:CD29hi) from lactation day 10 Ucp1-iCre-ROSAmTmG mice (n=2). GFP+ brown adipocytes from 8-week old female Myf5-Cre-ROSAmTmG mice (n=2). GFP+ brown adipocytes from 8-week old female Ucp1-iCre-ROSAmTmG mice (n=2).
Project description:Mature adipocytes from Inguinal WAT of CL316,243-treated Ucp1Cre/+;Rosa26-LSL-GFP mice were isolated and GFP+ adipocytes were collected and subjected to transcriptome profiling by RNA-seq.
Project description:We have identified a population of adipocytes in fat tissue that arise from bone marrow-derived progenitor cells. We used microarrays to compare the global gene expression patterns of the bone marrow progenitor-derived adipocytes as well as conventional white and brown adipocytes to evaluate the relationship between these adipocyte subpopulations. Gonadal fat tissue (for white adipocytes) and intrascapular fat tissue (for brown adipocytes) was digested with collagenase and adipocytes were recovered by centrifugation/flotation. Bone marrow derived adipocytes were isolated from the adipocyte fraction of gonadal fat tissue from mice receiving bone marrow tranplants from donors expressing either green fluorescent protein (GFP) or beta-galactosidase (LacZ) by flow cytometry.
Project description:Adipose tissue in the mammary gland undergoes dramatic remodeling during reproduction. Adipocytes are replaced by mammary alveolar structures during pregnancy and lactation, then reappear upon weaning. Here, we reveal that adipocytes in the mammary gland de-differentiate into Pdgfrα+ preadipocyte- and fibroblast-like cells during pregnancy, and remain de-differentiated during lactation. Upon weaning, de-differentiated fibroblasts proliferate and re-differentiate into adipocytes. In order to determine the molecular signature of these de-differentiated adipocytes in the mammary gland, we compared these cells with classical adipocytes. Using the AdipoChaser-mT/mG system, we pre-labeled mature adipocytes with GFP expression to characterize the features of these de-differentiated adipocytes (Figure 4A), and then purified CD31-/CD45-/PDGFRα+/Tomato+ and CD31-/CD45-/PDGFRα+/GFP+ cells from the stromal vascular fraction (SVF) of lactating mammary gland at the peak of lactation through FACS. Gene expression analyses showed that the CD31-/CD45-/PDGFRα+/Tomato+ cells were indeed enriched with Tomato expression, while the CD31-/CD45-/PDGFRα+/GFP+ cells were enriched with GFP expression (Figure 4C). We then collected CD31-/CD45-/PDGFRα+/GFP+ cells as single cells for subsequent single cell RNA-sequencing analysis (Figure 4D-G, Supplemental. Figure S1A-G). After the flow sorting and single cell RNA amplification, 26 CD31-/CD45-/PDGFRα+/GFP+ cells passed the quality control, and these cells were used for single-cell RNA-sequencing analysis. Due to technical difficulties in sorting single mature white adipocyte through flow cytometry, adipocytes differentiated from the immortalized murine-derived brown pre-adipocyte cell line were used as mature adipocyte control (Pradhan et al., 2017). Additionally, we also included population RNA-seq experiments, i.e. three mature white adipocyte samples, two GFP+, and six GFP- ones.
Project description:Brown adipocytes, muscle and dorsal dermis descend from precursor cells in the dermomyotome, but the factors that regulate commitment to the brown adipose lineage are unknown. Here, we prospectively isolated and determined the molecular profile of embryonic brown preadipose cells. Brown adipogenic precursor activity in embryos was confined to Pdgfrα+, Myf5Cre-lineage-marked cells. RNAseq analysis identified Early B Cell Factor-2 (Ebf2) as one of the most selectively expressed genes in this cell fraction. Importantly, Ebf2-expressing cells purified from Ebf2-GFP embryos or brown fat tissue did not express myoblast or dermal cell markers and uniformly differentiated into brown adipocytes. Interestingly, Ebf2-expressing cells from white fat tissue in adult animals differentiated into brown-like (or beige) adipocytes. Loss of Ebf2 in brown preadipose cells reduced the expression levels of brown preadipose-signature genes, whereas ectopic Ebf2-expression in myoblasts activated brown preadipose-specific genes. Altogether, these results indicate that Ebf2 specifically marks and regulates the molecular profile of brown preadipose cells. Embryonic fibroblasts, isolated from dorsal body wall of E14.5 Ebf2(GFP)/+ embryos, were further fractionated based on the expression of PDGFRα and Ebf2 (GFP). Affymetrix microarray analysis was prefromed,to compare the gene expression between PDGFRα+ Ebf2(GFP)- and PDGFRα+ Ebf2(GFP)+ cells.
Project description:We engineered KSR1-/- mouse embryonic fibroblasts (MEFs) to express GFP alone, KSR1 or RasV12 and GFP, or KSR1, H-RasV12 and GFP, and performed gene expression profiling on all 4 cell lines. Each cell line was measured in triplicate: 1. GFP alone 2. H-RasV12 and GFP 3. KSR1 and GFP 4. KSR1, H-RasV12, and GFP
Project description:Here, we report on experiments in double-transgenic mice, in which RFP is expressed in all Foxp3+ Treg cells, whereas Foxp3-dependent GFP expression is exclusively confined to intrathymically induced Foxp3+ Treg cells. This novel molecular genetic tool enabled us to faithfully track and characterize naturally induced Treg cells of intrathymic (RFP+GFP+) and extrathymic (RFP+GFP−) origin in otherwise unmanipulated mice. These experiments directly demonstrate that extrathymically induced Treg cells substantially contribute to the overall pool of mature Foxp3+ Treg cells residing in peripheral lymphoid tissues of steady-state mice. Furthermore, we provide evidence that intra- and extrathymically induced Foxp3+ Treg cells represent distinct phenotypic and functional sublineages. CD4+CD25+ RFP+GFP- and CD4+CD25+ RFP+GFP+ T cells from pooled lymph nodes and pooled spleens of 20 mice were FACS sorted for RNA extraction and hybridization on Affymetrix microarrays in duplicates.
Project description:Very little is known about how animals discriminate pathogens from innocuous microbes. To address this question, we examined infection-response gene induction in the nematode Caenorhabditis elegans. We focused on genes that are induced in C. elegans by infection with the bacterial pathogen Pseudomonas aeruginosa, but are not induced by an isogenic attenuated gacA mutant. Most of these genes are induced independently of known immunity pathways. We generated a GFP reporter for one of these genes, infection response gene 1 (irg-1), which is induced strongly by wild-type P. aeruginosa strain PA14, but not by other C. elegans pathogens or by other wild-type P. aeruginosa strains that are weakly pathogenic to C. elegans. To identify components of the pathway that induces irg-1 in response to infection, we performed an RNA interference screen of C. elegans transcription factors. This screen identified zip-2, a bZIP transcription factor that is required for inducing irg-1, as well as several other genes, and is important for defense against infection by P. aeruginosa. These data indicate that zip-2 is part of a specialized pathogen response pathway that is induced by virulent strains of P. aeruginosa and provides defense against this pathogen. Analysis of differential gene expression in adult N2 C. elegans treated with L4440 control RNAi or zip-2 RNAi, either uninfected (feeding on E. coli) or infected with P. aeruginosa PA14; samples were analyzed after 4 hours of infection