Project description:The aim of this study is to discover genes regulated by miR-204. Differential gene expression in HEK-293 cells transfected with miR-204-mimic compared to HEK-293 cells transfected with control oligo (HEK-293 control) was analyzed using the Agilent Human Whole Genome 4x44K gene expression array (Agilent Technologies, Santa Clara, CA). HEK-293 cells were transfected with either miR-204 or a control, and gene expression was analyzed using the Agilent Human Whole Genome 4x44K array. A dye-swap was performed.
Project description:To test the influence of IGF2BPs on the stability of their interacting mRNAs, as reported previously for some targets (Yisraeli, 2005), we simultaneously depleted all three IGF2BP family members using siRNAs and compared the cellular RNA from knockdown and mock-transfected cells on microarrays. The levels of transcripts identified by PAR-CLIP decreased in IGF2BP-depleted cells, indicating that IGF2BP proteins stabilize their target mRNAs. Moreover, transcripts that yielded clusters with the highest T to C mutation frequency were most destabilized (Figure 4G of PMID 20371350), indicating that the ranking criterion that we derived based on the analysis of PUM2 and QKI data generalizes to other RNA-binding proteins (RBPs). The RBPs IGF2BP1-3 were depleted by siRNAs and the expression level was compared to mock-transfected HEK 293 cells.
Project description:To assess whether the transcripts identified by PAR-CLIP are regulated by the RNA-binding protein (RBP) Quaking (QKI), we analyzed the mRNA levels of mock-transfected and QKI-specific siRNA-transfected cells with microarrays. Transcripts crosslinked to QKI were significantly upregulated upon siRNA transfection, indicating that QKI negatively regulates bound mRNAs (Figure 3H of PMID 20371350), consistent with previous reports of QKI being a repressor. The RBP QKI was depleted by siRNAs and the expression level was compared to mock-transfected HEK 293 cells.
Project description:To obtain evidence that Argonaute (AGO) crosslink-centered regions (CCRs) indeed contain functional miRNA-binding sites, we blocked 25 of the most abundant miRNAs in HEK 293 cells (Figure 5C of PMID 20371350) by transfection of a cocktail of 2'-O-methyl-modified antisense oligoribonucleotides and monitored the changes in mRNA stability by microarrays (Figure 7A of PMID 20371350). Consistent with previous studies of individual miRNAs (Grimson et al., 2007), the magnitude of the destabilization effects of transcripts containing at least one CCR depended on the length of the seed-complementary region and dropped from 9-mer to 8-mer to 7-mer to 6-mer matches (Figure 7B of PMID 20371350). We did not find evidence for significant destabilization of transcripts that only contained imperfectly paired seed regions. The top 25 expressed miRNAs expressed in HEK 293 cells were inhibited by transfection of a cocktail of 25 antisense 2'-O-Me oligoribonucleotides and the gene expression was compared to mock-transfected cells.
Project description:So far very little is known about the fine regulation of some of these intracellular interactions of β-DG and how they are perturbed in diseases. To start filling this gap, HEK-293 cells were transiently transfected with a plasmid carrying the β-DG subunit with GFP fused at its C-terminus. Immunoprecipitation by anti-GFP antibodies followed by shotgun proteomic analysis to investigate the proteins exclusively matching for β-DG binding, applying the following filters to MS identification data: high confidence, peptide rank 1, peptide length minimum 9 amino acid residues and selecting 2 peptides per protein. A series of already known β-DG interactors have been found, whilst significant new matches, which include potential novel β-DG interactors and their related networks, were identified in diverse subcellular compartments.
Project description:HEK 293 cells were transiently transfected with plasmids expressing Vector only(PCMV), Aire, or MBD-VP16 with the goal of comparing the global gene expression profiles in the Aire and MBD-VP16 groups We used microarrays to detail the global gene expression profile. HEK 293 cells were transiently transfected with plasmids expressing Vector only(PCMV), Aire, or MBD-VP16 and 72 hrs post transfection total RNA was collected for microarray analysis.
Project description:This SuperSeries is composed of the following subset Series: GSE17342: The role of miRNA in Wilms' tumorigenesis GSE28397: Copy number alteration in Wilms' tumor with custom-designed miRNA probes GSE28400: MIR-204 target gene Refer to individual Series
Project description:The aim of the project was to characterize changes in gene expression that are associated with induced autophagic flux. We engineered HeLa, HEK 293 and SH-SY5Y cell lines to express tandem-fluorecent LC3 (tf-LC3). The ratio of the red and green fluorophores indicated how much of the LC3 is in the acidic interior of lysosomes, which was a proxy measure for autophagic flux. RNA sequencing was performed for the cell lines at baseline and 1h, 15h and 30h after treatments. Additional untreated samples were also sequenced at some but not all time points. Autophagy was induced by amino acid starvation or mTOR inhibition (AZD8055).
Project description:Purpose: identify sites in endogenous mRNAs that are cut by KSHV SOX; Method: parallel analysis of RNA ends (PARE, following Zhai et al., 2014); Results: SOX cuts at discrete locations in mRNAs human Xrn1 was knocked down in HEK293T cells by shRNAs or siRNAs to stabilize degradation fragments with free 5' ends; GFP-SOX or GFP were transfected for ~24 hrs; total RNA samples were collected and subjected to PARE protocol (Zhai et al., 2014)