Project description:Copy number variation analysis between BJ cells and BJ cells derived iPS cells which were established by using non-transmissible measles virus vector DNA derived from BJ cell and iPS cells were extracted and analysis using CGH array
Project description:Skeletal muscle differentiation is a highly coordinated multistep process in which proliferating mononucleated myoblasts ?rst withdraw from the cell cycle, then differentiate into postmitotic mononucleated myocytes, and subsequently fuse into multinucleated myotubes which ?nally bundle to form mature muscle ?bers. All these processes are controlled by the sequential activation of myogenic regulatory factors, and especially MYOD1 is activated in the early phase to promote the transcription of muscle-specific genes coding for muscle proteins such as alpha-actin, myosin heavy chain and muscle creatine kinase. We used microarrays to detail the global gene expression changes associated with MYOD1 L122R mutation and identified MYOD1 L122R blocked myogenic differentiation and had a MYC-like transcriptional potential. wild type MYOD1, MYOD1 L122R, MYC, and GFP were introduced into BJ human fibroblast cells by retroviral infection. For retrovirus production, the pcx4 and pBabe vectors system were used. Retroviruses were obtained by using 293T cells as packaging cells, and were infected into BJ cell lines and selected with 500 ?g/ml Zeocin (Invitrogen, Carlsbad, CA, USA). After selection, RNAs were extracted and processed at MSKCC according to procedures recommended by Affymetrix (Santa Clara, CA, USA).
Project description:The goal of this project is to identify changes of cyto-nuclear import upon STING activation by cGAMP. BJ cells stably expressing TurboID-NES were treated with biotin to label cytoplasmic proteins, after which cells were further treated with or without cGAMP to activate STING signaling. Cells were then fractionated to collect the nuclei. The biotinylated proteins in the nuclear pellets were purified by streptavidin beads for mass spectrometry analysis.
Project description:Chromosomal instability in early cancer stages is caused by stress on DNA replication. The molecular basis for replication perturbation in this context is currently unknown. We studied the replication dynamics in cells in which a regulator of S-phase entry and cell proliferation, the Rb-E2F pathway, is aberrantly activated. Aberrant activation of this pathway by HPV-16 E6/E7 or cyclin E oncogenes, significantly decreased the cellular nucleotide levels in the newly transformed cells. Exogenously supplied nucleosides rescued the replication stress and DNA damage, and dramatically decreased oncogene-induced transformation. Increased transcription of nucleotide biosynthesis genes, mediated by expressing the transcription factor c-Myc, increased the nucleotide pool and also rescued the replication-induced DNA damage. Our results suggest a model for early oncogenesis in which uncoordinated activation of factors regulating cell proliferation leads to insufficient nucleotides that fail to support normal replication and genome stability. In order to understand the molecular basis for the low nucleotide pool in cells enforced to proliferate by oncogene expression, we performed unbiased whole transcriptome analysis of BJ cells in comparison to BJ cells expressing cyclin E, c-Myc and cells co-expressing both oncogenes. Our results suggest that Rb-E2F aberrant activation enforces cell proliferation but fails to activate the nucleotide biosynthesis pathway leading to insufficient pool of nucleotides required for normal replication. We analyzed two sets of BJ cells infected with empty pBabe, Cyclin E, c-Myc or coexpression of Cyclin E and c-Myc
Project description:We applied in parallel RNA-Seq and Ribosome-profiling analyses to immortalized human primary BJ fibroblast cells in which p53 was induced by Nutlin-3a RNA-seq, using Illumina HiSeq 2000, was applied to BJ cells treated with Nutlin-3a, at 5 timepoints: 0, 2, 4, 6, 19 hrs Ribosome profiling was applied to BJ cells treated with Nutlin-3a, at 5 timepoints: 0, 2, 4, 6, 19 hrs
Project description:Transcriptional profiling of human BJ fibroblasts comparing control FF shRNA expressing cells vs. BRD7 shRNA expressing cells under two conditions, either untreated or treated with 8uM nutln-3a for 8 hours. This experiment was done using two independent shRNAs targeting BRD7. Nutlin-3a was used to stabilize p53 and induce its transcriptional activity. Two-condition experiment, FF shRNA cells vs. BRD7 shRNAs cells in two experimental conditions, either untreated or treated with nutlin-3a.
Project description:Copy number variation analysis between BJ cells and BJ cells derived iPS cells which were established by using non-transmissible measles virus vector
Project description:Aim: identification of differentially expressed genes after LIN-9 depletion.<br> <br> hTERT immortalized human BJ fibroblasts were infected with pMSCV-Blasticidin based retroviruses encoding a shRNA which targets human LIN-9 and the empty vector respectively.<br> After 4 days of selection total RNA was isolated. Cy3 and Cy5 labelled cDNA probes were generated using the CyScribe Post-labelling Kit (Amersham Biosiences).