Project description:To gain insight into the function of both OCIAD paralogs, we used untargeted quantitative mass spectrometry to compare the whole-cell proteomes of control U2OS cells, U2OS cells with individual or double OCIAD1/OCIAD2 knockdown, and OCIAD1 knockdown cells in which OCIAD1 expression was reintroduced by lentiviral delivery.
Project description:Homologous recombination-mediated DNA repair deficiency (HRD) predisposes to cancer development, but also provides therapeutic opportunities. Here, we identified an HRD gene signature that robustly predicted HRD status. Unexpectedly, concurrent loss of PTEN in BRCA1-deficient cells might extensively rewire the HR repair network and confer resistance to PARP inhibitor, partially through over-expression of TTK. We used the HRD gene signature as a drug discovery tool and found several PARP-inhibitor-synergizing agents through the connectivity map. Thus gene expression profiling can be used to define the functional status of the HR repair network providing prognostic and therapeutic information. Various shRNAs that target genes involved in homologous recombination (HR) were transfected in MCF-10A non-transformed breast cells lines. Stable HR gene knockdown MCF-10A cells were seeded 200000 at 10 cm plate. Cells were harvested after 48 hours culturing and used for gene expression profiling. The shRNA that target CHK1 gene was transfected in human osteosarcoma U2OS cell line by lentiviral particles and selected stable CHK1 knockdown U2OS cells. Scrambled control shRNA-transfected U2OS cells were applying as control. Both stable CHK1 knockdown and control U2OS cells were seeded with 2 x 10^5 cells at 10 cm culture plate. Cells were cultured in McCOY 5A medium with 10% FBS and harvested after 48 hours culturing. mRNA was extracted from collected cells and performing gene expression profiling. Three biological replicates were applied.
Project description:We used GRO-seq to examine the effect of Myc activation on RNA transcription in U2OS cells. We measure in duplicates gene transcription rates in U2OS cells containing an inducible Myc expression vector that were induced or mock-treated in duplicates for 5 hours.
Project description:CRISPR screen: U2OS or U2OS p53KO cells expressing Cas9 were transduced with a whole-genome library of CRISPR sgRNAs, then treated with either DMSO or etoposide. Differential sgRNA abundances were calculated for each condition and used to determine the effect of each single gene knockout on fitness and the drug-induced death rate. RNA-Seq: U2OS or U2OS p53KO cells were cultured with either DMSO or etoposide for 48 hours, and then U2OS cells were incubated in this conditioned media for 8 hours. RNA was collected and use to observe differential expression changes between conditions.
Project description:We used RNA-seq to examine the effect of Myc activation on U2OS cells transcriptome. We also examined these effects in the presence of Torin-1, an inhibitor of mTOR We measure gene expression profiles in U2OS cells containing an inducible Myc expression vector that were induced or mock-treated in duplicates for 36 hours. In addition, we repeated the experiments in the presence of Torin-1, an inhibitor of mTOR.