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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression analysis of NT193 cells with an epithelial (E) and mesenchymal (M) phenotype

ABSTRACT: Cells have been collected by limited dilution and mRNA from 3 consequtive passages of cells cultured in Dulbecco’s modified Eagle’s medium (DMEM Dutscher L0104-500) containing 4.5 g/L glucose, L-glutamine, sodium pyruvate supplemented with 10% of inactivated fetal bovine serum (FBS, Dutscher S1510-500), penicillin (10 000 U/mL), streptomycin (10 mg/mL) (Dutscher P06-07100) were used for RNA seq analysis.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Mus musculus

SUBMITTER: Raphael Carapito

PROVIDER: E-MTAB-14200 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Tenascin-C shapes TRAIL control over tumor immunity

Project description:TRAIL cytotoxicity is exploited as anti-cancer therapy since many years, however incomplete knowledge is generating many obstacles. Here, we address whether the highly abundant extracellular matrix molecule tenascin-C (TNC) orchestrating an immune suppressive tumor microenvironment and binding TRAIL impacts TRAIL cytotoxicity. We used an immune competent autologous NT193 breast cancer model with a knockdown (KD) of Tnfsf10 (encoding TRAIL) and observed accelerated tumor growth marked by less apoptosis, more proliferation and less myeloid cell infiltration as seen by flow cytometry. Grafting the same Tnfsf10 KD cells into a TNC knockout host showed repressed tumor growth suggesting that TNC is involved in regulating TRAIL functions.

2024-05-30 | E-MTAB-14089 | biostudies-arrayexpress

Lymphopenia-induced T cell exhaustion in STING gain-of-function mouse model

Project description:STING gain-of-function (GOF) mutations lead to T cell lymphopenia, in the context of severe combined immunodeficiency (SCID) for STING GOF V154M mice. This T cell lymphopenia, which is of central origin, has been described as type I IFN independent and associated with dysfunctions of the rare mature T cells found in the periphery. To better describe the biological mechanisms of these dysfunctions, we performed a transcriptomic analysis by RNA-seq on sorted splenic CD4+ and CD8+ mature T cells from STING GOF mice. We highlighted an unexpected T cell exhaustion phenotype that could partly explain their dysfunctions. Acquired very early in life, but only once the peripheral environment is reached, the phenotype appeared to depend neither on type I IFNs, nor on the intrinsic activation of STING in T or stromal cells. Mechanistically, the few mature T cells reaching the periphery seem to be rapidly impacted by the lymphopenic environment through increased antigenic and IL-7 stimulations that could lead to their exhaustion. By using STING GOF long term-hematopoietic stem cells (LT-HSC) transplantations with supportive wild-type bone marrow (BM) cells, we prevented the T cell exhaustion of STING GOF T cells in the resulting non lymphopenic context. With the support of lymphopenic RAG1 hypomorphic mice developing the phenotype, our data uncover a lymphopenia-mediated T cell exhaustion mechanism in STING GOF mice, for which a synergistic effect of the mutation is also envisaged.

2024-01-07 | E-MTAB-13658 | biostudies-arrayexpress

Human transthyretin (TTR) gene expression is markedly increased in repair phenotype of hereditary ATTR Schwann cell model

Project description:TTR is markedly expressed in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (S-DMEM)-incubated TgS1 cells compared with Schwann cell medium (SCM)-incubated cells. c-Jun, Gdnf, and Sox2 were upregulated in S-DMEM-incubated TgS1 cell compared with SCM-incubated cells, while Mpz is suppressed in them.

2022-09-23 | GSE213537 | GEO

C2C12 Murine Myoblasts over-expressing Eda2r

Project description:C2C12 (ATCC, #CRL-1772), a myoblast cell line from the C3H mouse strain, was grown in DMEM (Dulbecco’s Modified Eagle’s Medium, Gibco), supplemented with 10% fetal bovine serum (FBS) (Euroclone, Pero, Italy) and 2 mM L-glutamine (Gibco), and maintained in culture at 37 °C with 5% CO₂, without reaching confluence to avoid fusion into myotubes. Transfection of murine myoblasts was performed using Lipofectamine. C2C12 myoblasts were grown in a 6-well plate and transfected with pCMX-GFP plasmid (gifted from Prof. Kakizuka, Japan) or EDA2R-pCMV6-AC-GFP-expressing plasmid (MG222258, Origene) using Lipofectamine 2000 (Life Technologies), according to the manufacturer’s directions. After 48 h, total RNA was isolated from cells with QIAzol Lysis Reagent (Qiagen) and extracted using miRNeasy Kit (Qiagen). RNA concentration was evaluated through Qubit RNA High Sensitivity Assay Kit (Invitrogen, Waltham, MA, USA) while RNA quality was established using 4200 Tapestation (Agilent Technologies, Santa Clara, CA, USA).

2023-09-01 | E-MTAB-13101 | biostudies-arrayexpress

Cadmium exposure induces SNAIL1 expression via downregulation of miR-30e in human bronchial epithelial cells

Project description:Purpose: To understand the molecular mechanisms underlying Cd exposure-induced diseases. Methods: Immortalized human bronchial epithelial cells (BEAS-2B) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Cellgro) supplemented with 1% Penicillin Streptomycin and 10% Fetal Bovine Serum (FBS, Atlanta Biologicals) at 37 degree C and 5 % CO2. For Cd exposure, 0, 2.5, 5 and 10 µM of CdCl2 was added to the media and the cells were cultured for 72h. Results: This study shows that cadmium exposure induces SNAIL1 expression via miR-30e downregulation and the cells undergo epithelial-mesenchymal transition.

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Real-time quantitative PCR analysis of UPR genes in human normal and osteoarthritic chondrocytes

Project description:Human chondrocytes were obtained from the cartilage of femoral condyles and tibial plateaus from individuals within 12 hours of death (normal) and from patients undergoing total knee arthroplasty (osteoarthritic, OA). The chondrocytes were released from the cartilage, seeded at high density, and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum and an antibiotic mixture (100 units/ml penicillin base and 100 μg/ml streptomycin base) at 37°C in a humidified atmosphere of 5% CO2/95% air. Total RNA was extracted from the primary chondrocytes and processed for qPCR according to the manufacturer's instructions (SA Biosciences RT2 Profiler PCR Array PAHS-089Z).

2015-10-06 | GSE73749 | GEO

Human Sumoylome (SUMO-2) upon bacterial infection

Project description:Given the role of SUMOylation in pathogenic infection, we wanted to evaluate the changes to the host SUMO-2 subproteome during Shigella infection. HeLa cells overexpressing TAP-SUMO-2 or TAP-empty were used in conjunction with SILAC (Stable Isotope Labeling of Amino acids in Culture) (Golebiowski et al, 2009, 2010). Cells were grown in Dulbecco’s modified Eagle’s medium except that L-arginine and L-lysine were replaced with stable isotope (SILAC) forms depending on the treatment. Medium was supplemented with 10% dialyzed fetal calf serum. SILAC experiment compared TAP-containing cells (Lys0 and Arg0) with invasive Shigella flexneri (M90T) infected TAP-SUMO-2-containing cells (4,4,5,5-D4-lysine, Lys4, and 13C6- arginine, Arg6) and TAP-SUMO-2-containing cells infected with (mxiD) non-invasive strain of Shigella (13C6 15N2-lysine, Lys8, and 13C6 15N4 -arginine, Arg10).

2018-10-19 | PXD001100 | Pride

Real-time quantitative PCR analysis of UPR genes in human osteoarthritic chondrocytes specimens treated with thapsigargin compared to the untreated (control) chondrocytes

Project description:Human osteoarthritic (OA) chondrocytes were obtained from the cartilage of femoral condyles and tibial plateaus from three patients undergoing total knee arthroplasty. The chondrocytes were released from the cartilage, seeded at high density, and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum and an antibiotic mixture (100 units/ml penicillin base and 100 μg/ml streptomycin base) at 37°C in a humidified atmosphere of 5% CO2/95% air. The chondrocytes from each of the three donors were left untreated (control) and treated with thapsigargin (50nM for 20hours).Total RNA was extracted from the chondrocytes and processed for qPCR according to the manufacturer's instructions (SA Biosciences RT2 Profiler PCR Array PAHS-089Z).

2015-10-06 | GSE73746 | GEO

Real-time quantitative PCR analysis of UPR genes in human osteoarthritic chondrocytes specimens treated with tunicamycin compared to the untreated (control) chondrocytes

Project description:Human osteoarthritic (OA) chondrocytes were obtained from the cartilage of femoral condyles and tibial plateaus from three patients undergoing total knee arthroplasty. The chondrocytes were released from the cartilage, seeded at high density, and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum and an antibiotic mixture (100 units/ml penicillin base and 100 μg/ml streptomycin base) at 37°C in a humidified atmosphere of 5% CO2/95% air. The chondrocytes from each of the three donors were left untreated (control) and treated with tunicamycin (500ng/ml for 20 hours).Total RNA was extracted from the chondrocytes and processed for qPCR according to the manufacturer's instructions (SA Biosciences RT2 Profiler PCR Array PAHS-089Z).

2015-10-06 | GSE73748 | GEO

Small-RNA high-throughput sequencing of head-and-neck cancer with or without human papilloma virus etiology

Project description:HPV positive head and neck cancer (HNC) are thought to have different etiology from HPV-unrelated HNC. There are indications that HPV positive HNC have better survival and that treatment options could be optimized in this set of tumours. In this study, we sought to evaluate miRNA profiles in the different groups of HNC based on cancer subsite (oral or oropharyngeal) and HPV presence (positive/negative; based on both viral DNA and RNA presence) all in comparison with normal tonsillar tissue from approximately aged subjects. From 61 enrolled patients, 22 were selected for NGS small RNA sequencing using Illumina small RNA library preparation kits and sequencing reagents on Nextseq 500 machine. Only samples with RIN >= 7 were included. manufacturers protocol was followed for library preparation. Indexing was done with indexes 1-22, and final libraries were normalizes to same amount after QBit measurement. Sequencing was done on Nextseq 500 machine and subsequent analyisis in Basespace (illumina). Raw fastq files were subsequently additionally trimmed of adapter sequences using FastQc Basespace app and differential expression evaluated with SmallRNA app. Customized plots were obtained after importing SmallRNA app calculated counts to DeSeq2 package in R and reanalysis therein.

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