Labelling and identification of proliferating cells is important for the study of physiological or pathological processes in high-content screening (HCS) assays. Here we describe ethynyl deoxyuridine (EdU) as a biomar...Labelling and identification of proliferating cells is important for the study of physiological or pathological processes in high-content screening (HCS) assays. Here we describe ethynyl deoxyuridine (EdU) as a biomarker for the assessment of cell proliferation and clearly demonstrate the feasibility of the EdU-labelling method for use in HCS assays. EdU detection is highly robust, reproducible, technically simple, and well suited for automated segmentation, which provides an excellent al- ternative for setting up multiplexed HCS assays of siRNA, miRNA and small-molecule libraries.展开更多
The therapeutic promise of small interfering RNAs （siRNAs） for specific gene silencing is dependent on the successful delivery of functional siRNAs to the cytoplasm. Their conjugation to an established delivery plat...The therapeutic promise of small interfering RNAs （siRNAs） for specific gene silencing is dependent on the successful delivery of functional siRNAs to the cytoplasm. Their conjugation to an established delivery platform, such as gold nanoparticles, offers tremendous potential for treating diseases and advancing our understanding of cellular processes. Their success or failure is dependent on both the uptake of the nanoparticles into the cells and subsequent intracellular release of the functional siRNA. In this study, utilizing gold nanoparticle siRNA-mediated delivery against C-MYC, we aimed to determine if we could achieve knockdown in a cancer cell line with low levels of intracellular glutathione, and determine the influence, if any, of polyethylene glycol （PEG） ligand density on knockdown, with a view to determining the optimal nanoparticle design to achieve C-MYC knockdown. We demonstrate that, regardless of the PEG density, knockdown in cells with relatively low glutathione levels can be achieved, as well as the possible effect of steric hindrance of PEG on the availability of the siRNA for cleavage in the intracellular environment. Gold nanoparticle uptake was demonstrated via transmission electron microscopy and mass spectroscopy, while knockdown was determined at the protein and physiological levels （cells in S-phase） by in-cell westerns and BrdU incorporation, respectively.展开更多
基金supported by the National Natural Science Foundation of China (30870535 and 90913017) (B. Zhang) Introduced Innovative R&D Team Program of Guangdong Province (Gene Silencing Technology and Therapeutics)
文摘Labelling and identification of proliferating cells is important for the study of physiological or pathological processes in high-content screening (HCS) assays. Here we describe ethynyl deoxyuridine (EdU) as a biomarker for the assessment of cell proliferation and clearly demonstrate the feasibility of the EdU-labelling method for use in HCS assays. EdU detection is highly robust, reproducible, technically simple, and well suited for automated segmentation, which provides an excellent al- ternative for setting up multiplexed HCS assays of siRNA, miRNA and small-molecule libraries.
文摘The therapeutic promise of small interfering RNAs （siRNAs） for specific gene silencing is dependent on the successful delivery of functional siRNAs to the cytoplasm. Their conjugation to an established delivery platform, such as gold nanoparticles, offers tremendous potential for treating diseases and advancing our understanding of cellular processes. Their success or failure is dependent on both the uptake of the nanoparticles into the cells and subsequent intracellular release of the functional siRNA. In this study, utilizing gold nanoparticle siRNA-mediated delivery against C-MYC, we aimed to determine if we could achieve knockdown in a cancer cell line with low levels of intracellular glutathione, and determine the influence, if any, of polyethylene glycol （PEG） ligand density on knockdown, with a view to determining the optimal nanoparticle design to achieve C-MYC knockdown. We demonstrate that, regardless of the PEG density, knockdown in cells with relatively low glutathione levels can be achieved, as well as the possible effect of steric hindrance of PEG on the availability of the siRNA for cleavage in the intracellular environment. Gold nanoparticle uptake was demonstrated via transmission electron microscopy and mass spectroscopy, while knockdown was determined at the protein and physiological levels （cells in S-phase） by in-cell westerns and BrdU incorporation, respectively.
