Unknown,Transcriptomics,Genomics,Proteomics

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Interaction effects of knockdown of PML and TNFalpha treatment in HUVEC cells

ABSTRACT: To study the interaction effects between promyelocytic leukemia (PML) gene knockdown and tumor necrosis factor-alpha (TNFalpha) signaling in human umbilical vein endothelial cells (HUVECs), we transfected control or two independent PML siRNAs into HUVEC cells without or with 20 ng/mL TNFalpha treatment for 20 h. The total RNA was extracted for gene expression microarray analyses. The experiment is a 3x2 two-factor design. One factor is siRNA and it has three levels (siCtrl, siPML1, siPML2). siCtrl is RISC-inducing non-targeting control siRNA. siPML1 and siPML2 designate two independent siRNAs against the PML gene. Two independent siRNAs were used to eliminate the off-target effects of siRNA. The other factor is TNFalpha treatment (20 ng/mL for 20 h) and it has two levels: Untreated (U) or Treated (T). Each sample has technical duplicates.

ORGANISM(S): Homo sapiens

SUBMITTER: Hung-Ying Kao

PROVIDER: E-GEOD-35000 | 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.