Project description:mESCs were cultured at least 2 weeks in medium medium: N2B27 for SILAC (DMEM/F12 for SILAC (AthenaES), Neurobasal for SILAC (AthenaES), Sodium Pyruvate (40 mg/mL), N2 (1X), B27 (0.5X), Pen/Strep (1%), L-glutamine (2 mM), beta-mercaptoethanol (50 µM)) + 2i/LIF supplemented with (medium) 13C6 15N4 L-arginine (0.65 mM), (medium) 13C6 L-arginine (0.55 mM), and L-Proline (200 mg/L)84. Before switching from light to heavy medium, cells were treated with 0.05 µg/mL CHX or equivalent dilution of DMSO in 6-well plates. We treated the cells with 0.05 µg/mL CHX. At timepoint 0 h, we replaced medium medium with pre-warmed heavy medium: N2B27 for SILAC (Thermo scientific) supplemented with (heavy) 13C6 15N2 L-Lysine-2HCl (0.65 mM), (heavy) 13C6 15N4 L-Arginine-HCl (0.55 mM), and L-Proline (200 mg/L) for mESC.
Project description:NIH/3T3 cells were cultured at least 2 weeks in light medium: DMEM for SILAC (Thermo scientific) supplemented with PS, dialyzed FBS (Thermo scientific), (light) L-Lysine-2HCl (0.666 mM), (light) L-Arginine-HCl (0.399 mM)44, and L-Proline (200 mg/L). Before switching from light to heavy medium, cells were treated with 0.05 µg/mL CHX or equivalent dilution of DMSO in 6-well plates. At timepoint 0 h, we replaced light medium with pre-warmed heavy medium: DMEM for SILAC (Thermo scientific) supplemented with PS, dialyzed FBS (Thermo scientific), (heavy) 13C6 15N2 L-Lysine-2HCl (0.666 mM), (heavy) 13C6 15N4 L-Arginine-HCl (0.399 mM), and L-Proline (200 mg/L) for NIH/3T3.
Project description:Myxoid liposarcoma (MLS) is the second most common type of liposarcoma, and today few model systems to study the disease exists. To be able to model the disease in vitro, cell-free scaffolds from MLS patient-derived xenograft (PDX) models were generated. The MLS scaffolds were then used as a 3D growth platform for MLS cell lines to study the cancer microenvironments impact on cellular heterogeneity using RNA sequencing. Key components in the microenvironment have also been shown to influence the fraction of cellular subpopulations, such as cancer stem cells and migratory cells but also to promote aggressive features of cancers. Therefore, to better understand and characterize these scaffolds, protein analyses were performed and links between scaffold compositions and the induced gene expression profiles of the cells grown therein could be made. This model system provides a better insight to the composition of the cell-free cancer microenvironment in a rare disease, which can lead to identification of novel malignancy inducing properties in MLS.
Project description:Telomere shortening can cause detrimental diseases and contribute to aging. It occurs due to the end replication problem in cells lacking telomerase. In addition, recent evidence revealed that telomere shortening can be attributed to difficulties of the semi-conservative DNA replication machinery to replicate through the bulk of telomeric DNA repeats. To investigate telomere replication in a comprehensive manner, we developed QTIP-iPOND, which enables purification of the proteins that associate with telomeres during their replication. We identify in addition to the core replisome a large number of proteins that specifically associate with telomere replication forks and validate their importance.
Project description:Non-coding variants coordinate transcription factor (TF) binding and chromatin mark enrichment changes over regions spanning >100 kb. We named these molecularly coordinated regions “variable chromatin modules” (VCMs), providing a conceptual framework of how regulatory variation might shape complex traits. To better understand the molecular mechanisms underlying VCM formation, here, we mechanistically dissect an uncharacterized VCM-modulating non-coding variant that is associated with reduced chronic lymphocytic leukemia (CLL) predisposition and disease progression. This common, germline variant constitutes a 5-bp indel that controls the activity of an AXIN2 gene-linked VCM by creating a MEF2 binding site, which, upon binding, activates a super-enhancer-like regulatory element. This triggers a big change in TF binding activity and chromatin state at an enhancer cluster spanning >150 kb, coinciding with long-range chromatin compaction and AXIN2 up-regulation. Our results support a model in which the indel acts as an AXIN2 VCM-activating TF nucleation event, which modulates CLL pathology.
Project description:Investigation of whole genome expression pattern of 60 and 72 hours post fertilization Danio Rerio embryos exposed to TMT and vehicle control Embryos were exposed to 10uM TMT or control from 48hpf to 60 or 72 hpf. Three replicates were collected for each time point. 40 embryos were pooled to comprise a replicate.
Project description:The rat pheochromocytoma cell line PC12 cells were cultured in complete DMEM till 80% confluence, then placed at 5000 cells per squared cm. Cells were then plated in 24-well plates for cell viability assay and in T75 flasks for RNA isolation. Medium was replaced with serum-free fresh medium for 12 hours prior to TMT treatment. Gene expression patterns were then analysed using Rat Expression Array 230A Experiment Overall Design: In this study we analize gene expression patterns in PC12 cells treated with Trimethyltin (TMT). We utilized control cells (untreated) and two different concentration (1 and 5) Experiment Overall Design: We used three biological replicates, for the three concentration tested, according to MIAME guidelines Experiment Overall Design: (total 9 chips were used in this study).
Project description:We performed DNA-protein interaction (ChIP-seq) analyses for Helicobacter pylori N6 wild-type (WT) and HP1021 deletion mutant (ΔHP1021::aphA-3) under oxidative stress (21% O2) and optimal microaerobic growth (5% O2) conditions. We detected 100 binding sites of HP1021 on the H. pylori N6 chromosome, most of which are promoter-located, likely affecting gene transcription. 84 of 100 identified HP1021 binding sites were located near promoter regions. EMSA and ChIP-qPCR confirmed the binding of HP1021 to the promoter region of a few genes.