By comparing HeLa cells lacking ATF7IP or SETDB1 generated through CRISPR/Cas9-mediated gene disruption to wild-type HeLa cells, the goal of the experiment was to determine the effect of loss of the SETDB1•ATF7IP complex on the transcriptome. Overall design: Total RNA-seq of three independent knockout HeLa clones lacking either ATF7IP or SETDB1
ATF7IP-Mediated Stabilization of the Histone Methyltransferase SETDB1 Is Essential for Heterochromatin Formation by the HUSH Complex.
Cell line, Subject
View SamplesHeLa cells lacking MORC2 generated through CRISPR/Cas9-mediated gene disruption were reconstituted with either wild-type or R252W mutant MORC2, and re-repression of HUSH target genes assessed by RNA-seq Overall design: Total RNA-seq of MORC2 knockout cells, either 1) mock transduced, 2) transduced with lentiviral vector encoding wild-type MORC2 or 3) transduced with lentviral vector encoding R252W MORC2.
Hyperactivation of HUSH complex function by Charcot-Marie-Tooth disease mutation in MORC2.
Cell line, Subject
View SamplesUnderstanding gene expression profile and transcriptional regulation of healthy adult human hepatocytes
Differentiation in stem/progenitor cells along fetal or adult hepatic stages requires transcriptional regulators independently of oscillations in microRNA expression.
Specimen part, Time
View SamplesFat tissue was resected during gastric bypass surgery for management of obesity. All subjects had fasted at least 10 hours before surgery. Subjects with malignancies were excluded. No subjects were taking thiazolidinediones or steroids. None had fasting plasma glucose levels over 120 mg/ dl. One half to 10 g of abdominal subcutaneous (external to the fascia superficialis), mesenteric, and greater omental fat were obtained from each subject. The tissue was collected in Hanks balanced salt solution with bicarbonate, penicillin, and gentamicin. Fat tissue was minced and then digested in HBSS containing 1 mg/ml collagenase and 7.5% fetal bovine serum in a 37*C shaking water bath until fragments were no longer visible and the digest had a milky appearance. Digests were filtered and centrifuged at 800xG for 10 min. The digests were treated with an erythrocyte lysis buffer. Cells were plated in 1:1 Dulbeccos modified Eagles medium:Hams F12 that contained 10% fetal bovine serum and antibiotics at a density of 4 x 104 cells/cm2. After 18 hours cultures were trypsinized until 95% of cells were detached (leaving endothelial cells and macrophages behind) and re-plated. Macrophages were rare (less than 5 per 106 cells, as assessed by phase contrast microscopy) in the re-plated cultures, irrespective of fat depot origin. Plating medium was changed every 2 days until confluence. For differentiation, preadipocytes were treated for 30 days with plating medium (without serum) enriched with 100 nM dexamethasone, 500 nM human insulin, 200 pM triiodothyronine, 0.5 *M rosiglitazone, antibiotics, and 540 *M methylisobutylxanthine (removed after 2 days). Higher rosiglitazone and insulin concentrations did not further enhance differentiation. Medium was changed every 2 days. For the final 2 days, differentiation medium was removed and cells were cultured in plating medium without serum. Undifferentiated preadipocytes were maintained in plating medium until confluence, when serum was removed for 2 days. For telomerase-expressing clones, preadipocytes were isolated and when cells had undergone 7 population doublings, they were transduced with a retrovirus containing the plasmid, pBABE-hTERT-Hygro. This vector expresses the human telomerase reverse transcriptase component (hTERT) driven by the Moloney murine leukemia virus long terminal repeat promoter and a hygromycin resistance sequence driven by the SV40 promoter. The 3 abdominal subcutaneous and 3 omental stably transduced, hygromycin-resistant clones capable of achieving confluence fastest were selected from 38 subcutaneous and 42 omental clones. Telomerase activity in these clones was verified using a PCR-based telomere repeat amplification protocol. RNA was isolated from preadipocytes by the Trizol method. RNA samples were labeled using the standard one-cycle Affymetrix GeneChip Eukaryotic Target Labeling Assay for Expression Analysis. Samples were hybridized for 16 hours at 45 C and 60 rpm, washed and stained according to the standard Affymetrix Antibody Amplification for Eukaryotic Targets protocol, and scanned at 488 nm. Images were quantified and linearly scaled using Affymetrix GeneChip Operating Software 1.1 using default analysis settings.
Identification of depot-specific human fat cell progenitors through distinct expression profiles and developmental gene patterns.
No sample metadata fields
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Loss of Lkb1 and Pten leads to lung squamous cell carcinoma with elevated PD-L1 expression.
Specimen part
View SamplesLung squamous cell carcinoma (SCC) is a deadly disease for which current treatments are inadequate. We demonstrate that bi-allelic inactivation of Lkb1 and Pten in the mouse lung led to SCC that recapitulated the histology, gene expression and microenvironment found in human disease. Lkb1/Pten-null (LP) tumors expressed the squamous markers Krt5, p63 and Sox2, and transcriptionally resembled the basal subtype of human SCC. In contrast to mouse adenocarcinomas, the LP tumors contained immune populations enriched for tumor-associated neutrophils. Sca1+/Ngfr+ fractions were enriched for tumor propagating cells (TPCs) that could serially transplant the disease in orthotopic assays. TPCs in the LP model and Ngfr+ cells in human SCCs highly expressed Pdl1, suggesting a novel mechanism of immune escape for TPCs.
Loss of Lkb1 and Pten leads to lung squamous cell carcinoma with elevated PD-L1 expression.
Specimen part
View SamplesLung squamous cell carcinoma (SCC) is a deadly disease for which current treatments are inadequate. We demonstrate that bi-allelic inactivation of Lkb1 and Pten in the mouse lung led to SCC that recapitulated the histology, gene expression and microenvironment found in human disease. Lkb1/Pten-null (LP) tumors expressed the squamous markers Krt5, p63 and Sox2, and transcriptionally resembled the basal subtype of human SCC. In contrast to mouse adenocarcinomas, the LP tumors contained immune populations enriched for tumor-associated neutrophils. Sca1+/Ngfr+ fractions were enriched for tumor propagating cells (TPCs) that could serially transplant the disease in orthotopic assays. TPCs in the LP model and Ngfr+ cells in human SCCs highly expressed Pdl1, suggesting a novel mechanism of immune escape for TPCs.
Loss of Lkb1 and Pten leads to lung squamous cell carcinoma with elevated PD-L1 expression.
Specimen part
View SamplesThe goal of this analysis was to assess the similarity in transcriptomes between WT and Coro1-/- across regulatory and conventional T cells. Overall design: mRNA profiles of wild-type and Coronin1A knockout from murine regulatory (trg) and conventional (con) T cells were generated by deep sequencing, in triplicate, using Illumina TruSeq stranded mRNA sample kit.
Disruption of Coronin 1 Signaling in T Cells Promotes Allograft Tolerance while Maintaining Anti-Pathogen Immunity.
Specimen part, Subject
View SamplesActivation of glycolytic genes by HIF-1 is considered critical for metabolic adaptation to hypoxia. We found that HIF-1 also actively suppresses glucose metabolism through the tricarboxylic acid cycle (TCA) by directly trans-activating the gene encoding pyruvate dehydrogenase kinase 1 (PDK1). PDK1 inactivates the TCA cycle enzyme, pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Forced PDK1 expression in hypoxic HIF-1-null cells increases ATP levels, attenuates hypoxic ROS generation and rescues these cells from hypoxia-induced apoptosis. These studies reveal a novel hypoxia-induced metabolic switch that shunts glucose metabolites from the mitochondria to glycolysis to maintain ATP production and to prevent toxic ROS production.
HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia.
No sample metadata fields
View SamplesAndrogens have been postulated to be important modulators of adipose tissue metabolism and fat cell function. In the present study, we investigated the response of male and female mice retroperitoneal adipose tissue to the non-aromatizable androgen dihydrotestosterone (DHT). Adipose tissue samples were obtained in gonadectomized (GDX) animals treated with vehicle (control group), or injected with 0.1mg DHT at 1, 3, 6, 12, 18 and 24h prior to necropsy. Transcripts which were significantly modulated were considered as androgen-responsive genes. Quantitative real-time RT-PCR was used to confirm results from the microarry analysis in a subset of 46 probe sets in male mice and 98 probe sets in female mice. Using both methods and considering peak time versus control, 74.5% and 61.2% of the modulated genes were confirmed by PCR in males and females, respectively. Four genes were significantly stimulated in a similar manner by DHT in both sexes, namely metallothionein 1 (Mt1), growth arrest and DNA-damage-inducible 45 gamma (Gadd45g), cyclin-dependent kinase inhibitor 1A (Cdkn1a), and fk506-binding protein 5 (Fkbp5). All these genes appear to be associated with a down-regulation of adipocyte differentiation/proliferation and adipogenesis. In conclusion, this study which evaluated the transcriptome response of adipose tissue to DHT in male and female mice suggests that DHT consistently modulates genes involved in the regulation of adipogenesis in retroperitoneal adipose tissue of both male and female animals.
Response of the adipose tissue transcriptome to dihydrotestosterone in mice.
No sample metadata fields
View Samples