Unknown,Transcriptomics,Genomics,Proteomics

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SiRNA knockdown of Sp3 in human

ABSTRACT: Multiple sclerosis (MS) is an inflammatory disease of the central nervous system and is generally considered to be autoimmune in nature. We previously demonstrated that the transcription factor Sp3 is significantly down-regulated in immune cells from MS patients. The potential role of Sp3 down-regulation in MS pathogenesis is not well understood. The function of endogenous Sp3 was assessed in vitro after siRNA-mediated knockdown of its transcript in Jurkat cells. Sp3 protein levels were reduced an average of 70%. ELISA studies demonstrated decreased endogenous production of IL-10 and TGFÎ²1 and increased endogenous production of TNFÎ± (p<0.05 in all assays). Subsequent microarray analysis demonstrated significantly altered expression of 36 genes (p<0.001 for each gene) compared with control samples. Analysis showed differential expression (p<0.005) of 8 gene pathways. Many of the genes and pathways that were regulated by Sp3 are involved in immune function, specifically with regard to apoptosis, cell-to-cell adhesion, integrin signaling, T-cell differentiation, and cytokine production. This study identifies mechanisms by which Sp3 may regulate immune function and suggests a basis for its potential contribution to MS disease pathogenesis. Experiment Overall Design: Two Sp3 siRNA knockdown groups (siRNA Sp3 #2: n=3, and #6: n=3) were clustered together into the Treated condition, and the two control groups (siRNA for GAPDH: n=3, and Non-transfected cells: n=3)were assembled to constitute the Control condition. Thus a total of 12 arrays (6 Controls and 6 treated) were used in this experiment.

ORGANISM(S): Homo sapiens

SUBMITTER: Azik Schwechter

PROVIDER: E-GEOD-7102 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:IL-33 is a nuclear cytokine from the IL-1 family that plays important roles in health and disease. Under healthy conditions, IL-33 is constitutively expressed to high levels in the nucleus of producing cells in various human and mouse tissues. The extracellular function of IL-33 cytokine has been well documented, but it remains unclear whether intracellular nuclear IL-33 has additional functions in the nucleus. Here, we used a global proteomic approach based on quantification of 5000 individual proteins by high-resolution mass spectrometry to compare the extracellular and intracellular roles of IL-33 in primary human endothelial cells, a major source of IL-33 protein in human tissues. Large-scale analysis of protein expression was performed either after stimulation of the cells with the IL-33 mature form IL-3395-270 (during 6h or 24h) or after siRNA knockdown of intracellular IL-33 (two experiments, each with a different pool of distinct siRNAs, noted siRNA1 and siRNA2). In each case, proteins were fractionated by 1D SDS-PAGE in 12 gel bands, and label-free quantitative analysis was performed. The present dataset contains the files for the two experiments of knockdown of endogenous nuclear IL-33 expression: - RNA silencing strategy 1. Knockdown of endogenous nuclear IL-33 expression was performed with a pool of four distinct siRNAs (Dharmacon ON-TARGETplus SMARTpool IL-33 siRNAs) that have been specifically modified for efficient silencing of the target gene with reduced off-target effects. Cells transfected with these siRNA duplexes (si1) were compared with those transfected with the provided controls (CTsi1). Three independent biological replicates (noted _A, _B, _C) were prepared and analyzed for each condition, leading to 6 different samples. Each of them was fractionated into 12 gel bands analyzed by nanoLC-MS/MS, leading to 72 raw files. - RNA silencing strategy 2. The second knockdown strategy was based on the use of an independent pool of three siRNAs targeting IL-33, predesigned by another provider using new and critical siRNA design rules (Sigma MISSION Predesigned Il-33 siRNAs based on Rosetta siRNA design algorithm). Cells transfected with these siRNA duplexes (si2) were compared with those transfected with the provided controls (CTsi2). Three independent biological replicates (noted _A, _B, _C) were prepared and analyzed for each condition, leading to 6 different samples. Each of them was fractionated into 12 gel bands analyzed by nanoLC-MS/MS, leading to 72 raw files.

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SiRNA knockdown of Sp3 in human

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