Effects of nicastrin (NCSTN) siRNA knockdown in HEK293 and HEK001 cells.
ABSTRACT: The goal of this study was to determine if knockdown of nicastrin induced a proinflammatory phenotype in HEK001 and HEK293 cells. Nicastrin (NCSTN) is a member of the gamma-secretase complex, and has been identified as the most frequently mutated gene in familial hidradenitis suppurativa. While much research has been done into the effects of PSEN1 and PSEN2 loss, less is known about isolated NCSTN haploinsufficiency. Two cell lines were knocked down with either NCSTN siRNA or an siRNA to luciferase in triplicate. RNA was extracted from drug selected knockdowns and profiled on the Illumina Human HT-12 v4 beadarray. Gene ontology analysis of differentially expressed genes revealed a proinflammatory and decreased proliferation signature in keratinocytes. HEK293 cells demonstrated expression signatures for decreased cholesterol synthesis and interferon-alpha signaling, as well as increased p53 signaling and caspase mediated cytoskeletal cleavage. 12 total samples. Two cell lines (HEK001 & HEK293) each with two treatments (NCSTN siRNA knockdown or pLKO luciferase knockdown) in three parallel 3 replicates each. For each line gives the changes specific to knockdown of gamma-secretase component nicastrin (NCSTN).
Project description:We studied the variations of mRNA amounts after Evi1 knockdown or Flag-Evi1 overexpression in SKOV-3 cells. Despites Evi1 discovery in 1988, its recognized role as a dominant oncogene in myeloid leukemia and more recently in epithelial cancers, only a few target genes were known and it was not clear why Evi1 was involved in cancer progression. Here we obtained the genomic binding occupancy and expression data for Evi1 in human ovarian carcinoma cells. We identified numerous Evi1 target cancer genes and genes controlling cell migration and adhesion. Moreover, we characterized a transcriptional cooperation between AP1 and Evi1 that regulated proliferation and adhesion through a feed-forward loop. Furthermore, this study provides human genome-wide mapping and downstream analyses for Evi1 that will be useful for the research community. 16 samples were collected. Each condition was done in 4 replicates, collected 65 hours after transfection. Transfections with control siRNA or Flag-expressing vector were used as controls.
Project description:To identify RNAs accumulating in P-bodies (cytoslic RNP granules), P-bodies were purified from HEK293 cell extracts using a Flow Activated Particle Sorting (FAPS) method. Sorted P-bodies were compared to the pre-sorted fraction to identify RNA enriched in P-bodies.
Project description:Double-stranded RNA (dsRNA) can enter different pathways in mammalian cells, including sequence-specific RNA interference, sequence-independent interferon response and editing by adenosine deaminases. To assess the potential of expressed dsRNA to induce interferon stimulated genes in somatic cells, we performed microarray analysis of HEK293 and HeLa cells transfected with a MosIR plasmid expressing an mRNA with a long inverted repeat structure in its 3’UTR (MosIR) or with a parental MosIR plasmid (without inverted repeat) as a control. Clustering analysis based on differentially expressed genes suggested that there was no common transcriptome signature in cells expressing dsRNA. Overall, the number of genes with altered expression upon transfection of the MosIR plasmid was rather small and only 19 probe sets, corresponding to 17 genes, were changed more than two-fold in both cell lines. Total RNA from cultured HEK293 or HeLa cells was used in each sample. Two independent biological replicates were analyzed for each condition.
Project description:We report that AUF1 modulates global mRNA stability and translation, in turn promoting the maintenance of DNA integrity. Please see individual series. For AUF1 PAR-CLIP, the four isoforms of AUF1 (p37, p40, p42, and p45) tagged with a Flag epitope were expressed in HEK293 cells. For total RNA-Seq HEK293 cells were transfected with Control siRNA, AUF1 siRNA, Empty Vector, Flag-AUF1 p37, p40, p42, or p45 as well as WI-38 cells were collected at PDL 15 and 55 and also transfected with Control siRNA, AUF1 siRNA, HuR siRNA. For Ribo-Seq HeLa cells were transfected with Control siRNA, AUF1 siRNA, or HuR siRNA.
Project description:We report that AUF1 modulates global mRNA stability and translation, in turn promoting the maintenance of DNA integrity. Please see individual series. In short, for AUF1 PAR-CLIP, the four isoforms of AUF1 (p37, p40, p42, and p45) tagged with a Flag epitope were expressed in HEK293 cells. For total RNA-Seq HEK293 cells were transfected with Control siRNA, AUF1 siRNA, Empty Vector, Flag-AUF1 p37, p40, p42, or p45 as well as WI-38 cells were collected at PDL 15 and 55 and also transfected with Control siRNA, AUF1 siRNA, HuR siRNA. For Ribo-Seq HeLa cells were transfected with Control siRNA, AUF1 siRNA, or HuR siRNA.
Project description:The HEK293 cells were transiently transfected by pDEST40-SOX11 or control plasmid (pDEST40).Cells were collected at 24 hours post-transfection.Total RNA from alll samples was extracted.Biotinylated cRNA was prepared using the Illumina total Prep RNA application Kits.Hybridization, washing and scanning were performed according to the Illumina BeadStation 500* manual (revision C)
Project description:This SuperSeries is composed of the following subset Series: GSE25210: Genome-wide mapping of Flag-Evi1 in SKOV-3 ovarian carcinoma cells GSE25212: Effects of Evi1 knockdown and overexpression in SKOV-3 ovarian carcinoma cells GSE33724: Effects of EVI1 mild expression in HeLa cells Refer to individual Series
Project description:Analysis of differentially expressed genes after siRNA mediated knock-down of the FET-family of proteins; the FUS, EWS, and TAF15 proteins. The FET-proteins are thought to function as transcription factors, and this hypothesis is tested in the present study. Results provide important information of the genes which expression are controlled by the FET-family of proteins. Total RNA obtained HEK293-cells transfected with siRNA targeted against the FUS, EWS, or TAF15 mRNA, or a combination of all three of them. Control cells are transfected with an equal amount of unspecific siRNA without known targets.
Project description:HEK293 cells were transfected with control plasmid (pcDNA1/Neo; Invitrogen) or with the plasmid encoding HCaRG by a standard calcium phosphate co-precipitation method. The clones expressing the highest levels of HCaRG, HCaRG clone 8 and 9 were used in this experiment, while clone transfected with vector alone, Neo clone, served as controls. Stable transfectants were synchronized and grown in the presence of 10% FBS for 48 h. Total RNAs were purified with the mini RNeasy kit (Qiagen).
Project description:To compare gene expression in human intestinal cell line after Sirt1 manipulation by overexpression or targetted siRNA knockdown Triplicate biological replicates for Caco-2 control and Sirt1 overexpression (6 samples), duplicate biological replicates for Caco-2 control siRNA and two non-overlapping targetted Sirt1 siRNAs (6 samples)