Project description:Notch signaling is critical for vascular morphogenesis by co-determining the sprouting behavior of endothelial cells. Here, we investigate the function of ubiquitin-specific peptidase 10 (USP10) in regulation of the turnover of the NOTCH1 intracellular domain. HUVEC were transfected with scrambled or USP10 targeting siRNA and then stimulated by treatment with DLL4 or control. RNA for analysis was isolated after 24 hours of DLL4 stimulation.
Project description:In the present study we implemented the bioUb approach to identify DUB substrates in a systemic manner. DUBs have been individually silenced so that the ubiquitination levels of their respective substrates are increased, and hence, upon isolation, susbtrates have been detected by mass spectrometry. Here we report quantitative proteomic data of the putative substrates of 5 human DUBs: USP1, USP7, USP9X, USP11 and USP42.
Project description:We identified Sox17 as a novel angiogenic transcription factor in the context of tumor. Our data revealed that Sox17 promotes tumor angiogenesis and tumor vessel abnormality. We found that Sox17 is specifically expressed in tumor endothelial cells within tumors. Our study elucidates a novel transcriptional regulation for tumor angiogenesis Control HUVECs vs. Sox17 knockdown HUVECs. Biological replicates: 3 control replicates, 3 transfected replicates.
Project description:Adenosine-to-inosine (A-to-I) RNA editing, which is catalyzed by a family of adenosine deaminase acting on RNA (ADAR) enzymes, is important in the epitranscriptomic regulation of RNA metabolism. However, the role of A-to-I RNA editing in vascular disease is unknown. Here we show that cathepsin S mRNA (CTSS), which encodes a cysteine protease associated with angiogenesis and atherosclerosis, is highly edited in human endothelial cells. The 3â?² untranslated region (3â?² UTR) of the CTSS transcript contains two inverted repeats, the AluJo and AluSx+ regions, which form a long stemâ??loop structure that is recognized by ADAR1 as a substrate for editing. RNA editing enables the recruitment of the stabilizing RNA-binding protein human antigen R (HuR; encoded by ELAVL1) to the 3â?² UTR of the CTSS transcript, thereby controlling CTSS mRNA stability and expression. In endothelial cells, ADAR1 overexpression or treatment of cells with hypoxia or with the inflammatory cytokines interferon-γ and tumor-necrosis-factor-α induces CTSS RNA editing and consequently increases cathepsin S expression. ADAR1 levels and the extent of CTSS RNA editing are associated with changes in cathepsin S levels in patients with atherosclerotic vascular diseases, including subclinical atherosclerosis, coronary artery disease, aortic aneurysms and advanced carotid atherosclerotic disease. These results reveal a previously unrecognized role of RNA editing in gene expression in human atherosclerotic vascular diseases. 1) Evaluation of transcriptome expression and RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in poly(A) RNA-seq data derived from endothelial cell transcriptome after ADAR1 or ADAR2 knockdown (n=2 biological replicates per condition, total n=8 biological samples). 2) Evaluation of transcriptome expression and RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in total-RNA-seq data derived from peripheral blood mononuclear cells (n=12 total biological samples; n=4 replicates per condition). 3) Evaluation of transcriptome expression and RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in total-RNA-seq data derived from endothelial cell transcriptome under basal and hypoxic conditions (n=2 biological replicates per condition, total n=4 biological samples). 4) Evaluation of RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in total RNA-seq data derived from endothelial cell transcriptome under basal and hypoxic conditions after ADAR1 knockdown (n=3 replicates per condition, total n=12 biological samples). 5) HuR iCLIP RNA-sequencing data derived from HUVEC HuR iCLIP after ADAR1 knockdown (scrambled control and siADAR1, n=1 per condition, total n=2 biological samples).
Project description:The tumor suppressor and deubiquitinase (DUB) BAP1 regulates chromatin-associated processes and is frequently mutated in various malignancies. BAP1 and its drosophila orthologue Calypso assemble DUB complexes with ASXL-1, -2, -3 paralogues and ASX respectively, and these cofactors are required for stimulating their DUB activity. However how the DUB activity of BAP1 is regulated remains largely unknown. Here we show that BAP1 promotes monoubiquitination of ASXLs on the ASXM/DEUBAD domain. ASXL2 monoubiquitination promotes its stability or proteasomal degradation, stimulates BAP1 DUB activity and is required for mammalian cell proliferation. Monoubiquitination of ASXL2 is directly catalyzed by UBE2E family of ubiquitin conjugating enzymes and is regulated by deubiquitination. Monoubiquitination of ASX is regulated by Calypso and is required for drosophila development. We further revealed a switch mechanism that tightly regulate BAP1 function, as a monoubiquitination of BAP1 UCH domain is mutually exclusive with ASXL2 monoubiquitination, thus ensuring highly coordinated DUB-mediated signaling.
Project description:p/CIP binds to many nuclear receptors and plays a major role in hormone dependent transcription of genes. Recently, p/CIP was shown to affect mouse stem cell pluripotency. Microarray gene expression analysis was conducted to assess the role of p/CIP in mouse embryonic stem cells. p/CIP was downregulated using two different siRNAs and total RNA was extracted and hybridized to a microarray. As control, wild type cells were transfected with pooled non specific siRNAs. The expression difference was calculated between the control wild-type cells and cells having p/CIP downregulation. The experiments were carried out at London Regional Genomic Centre (http://www.lrgc.ca/).
Project description:To identify genes regulated by NFE2L2 (Nrf2), we selected a lung cancer cell line (A549) in which NFE2L2 is normally active. Three transfections using siRNAs targeting NFE2L2 and four control transfections using two different negative control siRNAs were done. As a result, we found several genes up or down regulated in response to NFE2L2 inactivation in these cells. A549 cells were cultured, and 250,000 cells were transfected with Lipofectamine RNAiMAX reagent containing 5 nmol siRNA particles in a 6-well plate. Three transfections with siRNAs targeting NFE2L2 were performed, using three independent duplexes, while four control transfections were done using two different control duplexes, in addition to one mock (Lipofectamine only) transfection. RNA was prepared using the RNeasy Mini kit. 8 strand-specific total RNA-seq libraries were generated on an Illumina NextSeq 500. Between 50.5 and 60.6 million reads were obtained per sample. Reads were aligned to hg19 genome, at an alignment rate exceeding 94.4% for all samples.
Project description:KLF2 and KLF4 are important transcriptional factors in endothelial cells, however their roles in statin treatment has not been elucidated. Here we report the comprehensive change of transcripts of statin treated HUVECs transfected with siRNA KLF2 or KLF4. We used repeated microarray analysis of HUVECs treated with pitavastatin for 4hours. Before statin treatment, cells were transfected with siRNA KLF2 or KLF4. HUVECs were used within the first 6 passages. For studies, HUVECs were cultivated in medium EGM2MV containing pitavastatin at a concentration of 1 micromolar.
Project description:Vascularization represents an important issue in bone development, fracture healing and engineering of artificial bone tissue. In the context of bone tissue engineering, it was shown that coimplantation of human primary umbilical vein endothelial cells (HUVECs) and human osteoblasts (hOBs) results in the formation of functional blood vessels and enhanced bone regeneration. Implanted endothelial cells do not only contribute to blood vessel formation, but also support proliferation, cell survival and osteogenic differentiation of coimplanted hOBs. These effects are partially mediated by direct heterotypic cell contacts. In a previous report we could show that cocultivated hOBs strongly increase the expression of genes involved in extracellular matrix (ECM) formation in HUVECs, suggesting that ECM may be involved in the intercellular communication between hOBs and HUVECs. The present study aimed at investigating whether comparable changes occur in hOBs. We therefore performed a microarray analysis of hOBs cultivated in direct contact with HUVECs, revealing 1121 differentially expressed genes. The differentially expressed genes could be assigned to the functional clusters ECM, proliferation, apoptosis and osteogenic differentiation. In summary, our data demonstrate that HUVECs provoke complex changes in gene expression patterns in cocultivated hOBs and that ECM plays and important role in this interaction.