Isolation of endogenous cytosolic DNA from cultured cells

Mosley, Shayla R.; Baker, Kristi

doi:10.1016/j.xpro.2022.101165

Cited by 5 publications

(3 citation statements)

References 9 publications

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“…Membranes were incubated in primary antibody overnight at 4°C followed by HRP‐conjugated secondary antibodies (1:5000; Cell Signaling) for 1 h at room temperature and then 1 min in ECL reagent (ThermoFisher/Pierce) for imaging on a ProteinSimple Alpha Imager. Cytoplasmic DNA was measured by lysing and fractionating remaining samples according to standardized protocols (Mosley & Baker, 2022 ; Spada et al, 2019 ) with a cytosolic‐nuclear fractionation kit optimized for frozen tissues (Invent Biotechnologies). Protein concentration was determined for each fraction using a BCA kit.…”

Section: Methodsmentioning

confidence: 99%

“…Gene Ontology analyses (Biological Processes/KEGG pathways) were performed using g:Profiler (Raudvere et al, 2019). to standardized protocols (Mosley & Baker, 2022;Spada et al, 2019) with a cytosolic-nuclear fractionation kit optimized for frozen tissues (Invent Biotechnologies). Protein concentration was determined for each fraction using a BCA kit.…”

Section: Rna-seq and Transcriptome Analyses On Mouse Tissuementioning

confidence: 99%

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The reverse transcriptase inhibitor 3TC protects against age‐related cognitive dysfunction

et al. 2023

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Aging is the primary risk factor for most neurodegenerative diseases, including Alzheimer's disease. Major hallmarks of brain aging include neuroinflammation/immune activation and reduced neuronal health/function. These processes contribute to cognitive dysfunction (a key risk factor for Alzheimer's disease), but their upstream causes are incompletely understood. Age‐related increases in transposable element (TE) transcripts might contribute to reduced cognitive function with brain aging, as the reverse transcriptase inhibitor 3TC reduces inflammation in peripheral tissues and TE transcripts have been linked with tau pathology in Alzheimer's disease. However, the effects of 3TC on cognitive function with aging have not been investigated. Here, in support of a role for TE transcripts in brain aging/cognitive decline, we show that 3TC: (a) improves cognitive function and reduces neuroinflammation in old wild‐type mice; (b) preserves neuronal health with aging in mice and Caenorhabditis elegans; and (c) enhances cognitive function in a mouse model of tauopathy. We also provide insight on potential underlying mechanisms, as well as evidence of translational relevance for these observations by showing that TE transcripts accumulate with brain aging in humans, and that these age‐related increases intersect with those observed in Alzheimer's disease. Collectively, our results suggest that TE transcript accumulation during aging may contribute to cognitive decline and neurodegeneration, and that targeting these events with reverse transcriptase inhibitors like 3TC could be a viable therapeutic strategy.

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Section: Methodsmentioning

confidence: 99%

Section: Rna-seq and Transcriptome Analyses On Mouse Tissuementioning

confidence: 99%

The reverse transcriptase inhibitor 3TC protects against age‐related cognitive dysfunction

et al. 2023

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“…CyDNA Isola on. CyDNA was isolated as previously described 54 . Briefly, 5x10 6 MC38 cells were collected by trypsiniza on before extrac on of the cytosolic frac on by incuba on in cytosolic extrac on buffer (150mM NaCl, 50mM HEPES, 200µg/mL Digitonin, 1M Hexylene Glycol) for 10 minutes on ice and centrifuga on at 2000g for 10 minutes at 4°C.…”

Section: Discussionmentioning

confidence: 99%

Mechanisms of genomic instability dictate cytosolic DNA composition and dendritic cell mediated anti-tumor immunity

Mosley,

Chen,

Doell

et al. 2024

Preprint

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Patients with microsatellite instable (MSI) colorectal cancers (CRC) face better prognosis than those with the more common chromosomal instable (CIN) subtype due to improved anti-tumor immune responses characterized by high cytotoxic T cell infiltration. Previous investigation identified the cytosolic DNA (cyDNA) sensor STING as necessary for chemokine-mediated T cell recruitment in MSI CRCs. Here, we find cyDNA from MSI CRC cells is inherently more capable of inducing STING activation and induces improved cytotoxic T cell activation by dendritic cells (DCs). Sequencing indicates MSI cyDNA is enriched for microsatellites, which upon DC uptake induce anti-tumor immunity in a manner consistent with clinical MSI CRCs. Radiation also modulates cyDNA stimulation capacity through larger cyDNA size and increased mitochondrial DNA content. Identifying highly stimulatory cyDNA arising from genomic instability such as in MSI CRCs allows for optimized development of DNA-based STING agonist therapies to improve responses of CIN CRC patients to immunotherapies.

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Immune modulation of the liver metastatic colorectal cancer microenvironment via the oral CAPOX-mediated cGAS-STING pathway

Park,

Kweon,

Moyo

et al. 2024

Biomaterials

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Isolation of endogenous cytosolic DNA from cultured cells

Cited by 5 publications

References 9 publications

The reverse transcriptase inhibitor 3TC protects against age‐related cognitive dysfunction

The reverse transcriptase inhibitor 3TC protects against age‐related cognitive dysfunction

Mechanisms of genomic instability dictate cytosolic DNA composition and dendritic cell mediated anti-tumor immunity

Immune modulation of the liver metastatic colorectal cancer microenvironment via the oral CAPOX-mediated cGAS-STING pathway

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