Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Modulation of EDA2R/EDA-A2 signalling in Human Myoblasts

ABSTRACT: Transfection of human myoblasts was performed using Lipofectamine or electroporation. Skeletal muscle myoblasts isolated from a human male donor were transfected using the Lipofectamine™ 2000 Transfection Reagent (Invitrogen, 11668019). Twenty-four hours before transfections, cells were plated to a density of 17,500 cells/cm² in a 6-well plate. The transfection was performed according to the manufacturer’s protocol. For each well, 4 µg of plasmid DNA (EDA2R, Origene) and the appropriate amount of Lipofectamine™ 2000 were diluted in Opti-MEM™ I Reduced Serum Medium (Gibco, 31985070). After transfection, cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 24 hours. For electroporation, skeletal muscle myoblasts isolated from a human male donor were transfected using the Amaxa™ Nucleofector Basic Kit for Primary Mammalian Smooth Muscle Cells (Lonza, VPI-1004). For each transfection condition, a total of 1 million cells were resuspended in the Nucleofector Solution provided with the kit. Then, 3 µg of plasmid DNA (EDA2R, Origene) was added to the cell suspension and subjected to nucleofection using the Amaxa Nucleofector Device with the A-033 program, as recommended by the manufacturer’s protocol. Following nucleofection, the cells were immediately transferred into pre-warmed complete growth medium and distributed into 3 wells of 9.6 cm² each. The cells were then incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 48 hours, until RNA extraction. For the ligand-based treatment, skeletal muscle myoblasts isolated from a human male donor were treated with EDA-A2 ligand (922-ED, R&D Systems) at the concentration of 250 ng/ml used in Bilgic et al., and incubated for an additional 48 hours, until RNA extraction.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Homo sapiens

SUBMITTER: Luca Guarrera

PROVIDER: E-MTAB-14239 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:SF-1 (NR5A1) was overexpressed (Over) or knocked down with shRNA (shRNA) in NCI-H295R human adrenocortical tumor cells and differential global gene expression analysed 48 hours later using Affymetrix GeneChip Human Gene 1.0ST arrays. Over: 5 million cells were transfected (Amaxa Nucleofection) with 10 ug of a pIRES2-AcGPF1-Nuc construct co-expressing SF-1 cDNA and GFP. For experimental control, a mutagenized pIRES2 construct, bearing the G35E mutation in SF-1 that impairs its transactivation function in vivo and in vitro was used. shRNA: 5 million cells were transfected (Amaxa Nucleofection) with 10 ug of the SureSilencing shRNA Plasmid for Human NR5A1 with GFP marker kit (SABioscience). For experimental control, mismatch constructs provided in the kit were used. In both experiments (Over and shRNA), cells were harvested, prepared, and submitted to fluorescence-activated cell sorting (FACS) in a MoFlo XDP sorter 48 hours after transfection. Viable GFP-expressing cells were pooled and resuspended in TRIzol reagent for RNA extraction. Total RNA was extracted, and RNA quality control performed using a 2100 Bioanalyzer. RNA samples were processed using the Affymetrix GeneChip WT Sense Target Labeling kit, starting with 200 ng total RNA. Four independent Over experiments and five independent shRNA experiments were performed and samples of labeled fragmented cDNA were hybridized to Affymetrix GeneChip Human Gene 1.0 ST Arrays. Based on quality control of array data (R/Bioconductor and Partek Genomics Suite), two Over arrays (one SF-1 wild type and one mutant control) and one shRNA array (mismatch control) were excluded and are not deposited here. Differential gene expression analysis was performed using the limma package in R/Bioconductor. A Benjamini-Hochberg-corrected P value cut-off of 0.05 was used to select significant differentially expressed genes in each setting (Over and shRNA). Finally, results from both analyses were combined to identify a subset of positively-regulated SF-1 target genes in these cells, ie transcripts up-regulated by SF-1 overexpression (Over) and down-regulated by SF-1 knockdown (shRNA).

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Modulation of EDA2R/EDA-A2 signalling in Human Myoblasts

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