Nuclei Isolation Staining (NIS) Method for Imaging Chromatin‐Associated Proteins in Difficult Cell Types

Neely, Amy E.; Bao, Xiaomin

doi:10.1002/cpcb.94

Cited by 6 publications

(3 citation statements)

References 17 publications

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“…To test this, we applied Stochastic Optical Reconstruction Microscopy (STORM), with spatial resolution at ~25 nm, to resolve individual NPCs and enable quantification of endogenous NUP proteins. As STORM imaging relies on high-quality antibody binding, we implemented the Nuclei-Isolation Staining (NIS) method 27 to enhance antibody nuclear accessibility. NIS reduces STORM imaging background and enhances imaging quality, by removing cytoplasmic proteins that can contribute to non-specific antibody binding.…”

Section: Resultsmentioning

confidence: 99%

Nucleoporin downregulation modulates progenitor differentiation independent of nuclear pore numbers

Neely,

Zhang,

Blumensaadt

et al. 2023

Commun Biol

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Nucleoporins (NUPs) comprise nuclear pore complexes, gateways for nucleocytoplasmic transport. As primary human keratinocytes switch from the progenitor state towards differentiation, most NUPs are strongly downregulated, with NUP93 being the most downregulated NUP in this process. To determine if this NUP downregulation is accompanied by a reduction in nuclear pore numbers, we leveraged Stochastic Optical Reconstruction Microscopy. No significant changes in nuclear pore numbers were detected using three independent NUP antibodies; however, NUP reduction in other subcellular compartments such as the cytoplasm was identified. To investigate how NUP reduction influences keratinocyte differentiation, we knocked down NUP93 in keratinocytes in the progenitor-state culture condition. NUP93 knockdown diminished keratinocytes’ clonogenicity and epidermal regenerative capacity, without drastically affecting nuclear pore numbers or permeability. Using transcriptome profiling, we identified that NUP93 knockdown induces differentiation genes related to both mechanical and immune barrier functions, including the activation of known NF-κB target genes. Consistently, keratinocytes with NUP93 knockdown exhibited increased nuclear localization of the NF-κB p65/p50 transcription factors, and increased NF-κB reporter activity. Taken together, these findings highlight the gene regulatory roles contributed by differential NUP expression levels in keratinocyte differentiation, independent of nuclear pore numbers.

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Section: Resultsmentioning

confidence: 99%

Nucleoporin downregulation modulates progenitor differentiation independent of nuclear pore numbers

Neely,

Zhang,

Blumensaadt

et al. 2023

Commun Biol

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“…The nuclei were pelleted by centrifugation at 500 x g for 10 min. The pellet was resuspended in 4 mL nuclei extraction buffer (10 mM Hepes pH 7.4, 1.5 mM MgCl 2 , 10 mM KCl, 1X protease inhibitor cocktail and 0.2% NP40 substitute) ( Neely and Bao, 2019 ). The samples were split into 1 mL aliquots and added to poly-L-ornithine-treated (Millipore Sigma) glass cover slips (Thermo Fisher Scientific) by centrifugation at 500 x g at 4°C in a 12 well cell culture plate.…”

Section: Methodsmentioning

confidence: 99%

Improved metagenome assemblies through selective enrichment of bacterial genomic DNA from eukaryotic host genomic DNA using ATAC-seq

Cantin,

Dunning Hotopp,

Foster

2024

Front. Microbiol.

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Genomics can be used to study the complex relationships between hosts and their microbiota. Many bacteria cannot be cultured in the laboratory, making it difficult to obtain adequate amounts of bacterial DNA and to limit host DNA contamination for the construction of metagenome-assembled genomes (MAGs). For example, Wolbachia is a genus of exclusively obligate intracellular bacteria that live in a wide range of arthropods and some nematodes. While Wolbachia endosymbionts are frequently described as facultative reproductive parasites in arthropods, the bacteria are obligate mutualistic endosymbionts of filarial worms. Here, we achieve 50-fold enrichment of bacterial sequences using ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) with Brugia malayi nematodes, containing Wolbachia (wBm). ATAC-seq uses the Tn5 transposase to cut and attach Illumina sequencing adapters to accessible DNA lacking histones, typically thought to be open chromatin. Bacterial and mitochondrial DNA in the lysates are also cut preferentially since they lack histones, leading to the enrichment of these sequences. The benefits of this include minimal tissue input (<1 mg of tissue), a quick protocol (<4 h), low sequencing costs, less bias, correct assembly of lateral gene transfers and no prior sequence knowledge required. We assembled the wBm genome with as few as 1 million Illumina short paired-end reads with >97% coverage of the published genome, compared to only 12% coverage with the standard gDNA libraries. We found significant bacterial sequence enrichment that facilitated genome assembly in previously published ATAC-seq data sets from human cells infected with Mycobacterium tuberculosis and C. elegans contaminated with their food source, the OP50 strain of E. coli. These results demonstrate the feasibility and benefits of using ATAC-seq to easily obtain bacterial genomes to aid in symbiosis, infectious disease, and microbiome research.

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“…The cells were plated in a T25 flask and treated with vehicle (DMSO) or doxycycline for 48hrs. Cells were harvested, and nuclei were isolated using hypotonic buffer as previously described (98). The isolated nuclei were spun down onto the coverslip precultured with poly-L-ornithine (Sigma) at 400G for 5 mins, then fixed by 4% paraformaldehyde at room temperature for 10 mins, following 3 times washing with PBS.…”

Section: Immunofluorescence (If) Microscopymentioning

confidence: 99%

The cytidine deaminase APOBEC3G drives cancer mutagenesis and clonal evolution in bladder cancer

Liu

Newhall

Khani

et al. 2022

Preprint

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Mutagenic processes leave distinct signatures in cancer genomes. The mutational signatures attributed to APOBEC3 cytidine deaminases are pervasive in human cancers. However, data linking individual APOBEC3 proteins to cancer mutagenesis in vivo are limited. Here, we show that transgenic expression of human APOBEC3G promotes mutagenesis, genomic instability, and kataegis, leading to shorter survival in a murine bladder cancer model. Acting as mutagenic fuel, APOBEC3G increases the clonal diversity of bladder cancers, driving divergent cancer evolution. We characterize the single base substitution signature induced by APOBEC3G in vivo, showing the induction of a mutational signature different from that caused by APOBEC3A and APOBEC3B. Analysis of thousands of human cancers reveals the contribution of APOBEC3G to the mutational profiles of multiple cancer types, including bladder cancer. Our findings define the role of APOBEC3G in cancer mutagenesis and clonal heterogeneity. These results potentially inform future therapeutic efforts that restrict tumor evolution.

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Nuclei Isolation Staining (NIS) Method for Imaging Chromatin‐Associated Proteins in Difficult Cell Types

Cited by 6 publications

References 17 publications

Nucleoporin downregulation modulates progenitor differentiation independent of nuclear pore numbers

Nucleoporin downregulation modulates progenitor differentiation independent of nuclear pore numbers

Improved metagenome assemblies through selective enrichment of bacterial genomic DNA from eukaryotic host genomic DNA using ATAC-seq

The cytidine deaminase APOBEC3G drives cancer mutagenesis and clonal evolution in bladder cancer

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