ALT-FISH quantifies alternative lengthening of telomeres activity by imaging of single-stranded repeats DOI Creative Commons
Lukas Frank, Anne Rademacher, Norbert Mücke

et al.

Nucleic Acids Research, Journal Year: 2022, Volume and Issue: 50(11), P. e61 - e61

Published: Feb. 5, 2022

Alternative lengthening of telomeres (ALT) occurs in ∼10% cancer entities. However, little is known about the heterogeneity ALT activity since robust detection assays with high-throughput situ readouts are lacking. Here, we introduce ALT-FISH, a method to quantitate single cells from accumulation single-stranded telomeric DNA and RNA. It involves one-step fluorescent hybridization approach followed by fluorescence microscopy imaging. Our reliably identified cell lines different tumor entities was validated three established models induction suppression. Furthermore, successfully applied ALT-FISH spatially resolve primary tissue sections leiomyosarcoma neuroblastoma tumors. Thus, our assay provides insights into tumors suited for applications, which will facilitate screening ALT-specific drugs.

Language: Английский

EASI-ORC: A pipeline for the efficient analysis and segmentation of smFISH images for organelle-RNA colocalization measurements in yeast DOI Creative Commons
Shahar Garin,

Liav Levavi,

Jeffrey E. Gerst

et al.

Communications Biology, Journal Year: 2025, Volume and Issue: 8(1)

Published: Feb. 15, 2025

Analysis of single-molecule fluorescent in situ hybridization (smFISH) images is important to translate cellular image data into a quantifiable format. Although smFISH the gold standard for RNA localization measurements, there are no freely available, user-friendly applications assaying messenger (mRNA) organelles. EASI-ORC (Efficient and Segmentation Images Organelle-RNA Colocalization) novel pipeline automated analysis multiple yeast cells. automates segmentation cells organelles, identifies bona fide signals, measures mRNA-organelle colocalization. efficient, unbiased, plots colocalization statistical analyses. utilizes existing ImageJ plugins original scripts, thus allowing free access ease-of-use. To circumvent technical literacy issues, step-by-step user guide provided. offers robust solution - one that saves time, effort provides consistent measurements yeast.

Language: Английский

Citations

0
Deep-tissue transcriptomics and subcellular imaging at high spatial resolution DOI
Valentina Gandin, Jun Kim,

Liang-Zhong Yang

et al.

Science, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 20, 2025

Limited color channels in fluorescence microscopy have long constrained spatial analysis biological specimens. Here, we introduce cycle Hybridization Chain Reaction (HCR), a method that integrates multicycle DNA barcoding with HCR to overcome this limitation. cycleHCR enables highly multiplexed imaging of RNA and proteins using unified barcode system. Whole-embryo transcriptomics achieved precise three-dimensional gene expression cell fate mapping across specimen depth ~310 μm. When combined expansion microscopy, revealed an intricate network 10 subcellular structures mouse embryonic fibroblasts. In hippocampal slices, multiplex protein uncovered complex gradients cell-type-specific nuclear structural variations. provides quantitative framework for elucidating regulation deep tissue contexts research potentially diagnostic applications.

Language: Английский

Citations

0
Functional genomic profiling of O-GlcNAc reveals its context-specific interplay with RNA polymerase II DOI Creative Commons
Sofia Rucli, Nicolas Descostes, Yulia G. Ermakova

et al.

Genome biology, Journal Year: 2025, Volume and Issue: 26(1)

Published: March 24, 2025

Abstract Background How reversible glycosylation of DNA-bound proteins acts on transcription remains scarcely understood. O-linked β-N-acetylglucosamine (O-GlcNAc) is the only known form modifying nuclear proteins, including RNA polymerase II (RNA Pol II) and many factors. Yet, regulatory function O-GlcNAc modification in mammalian chromatin unclear. Results Here, we combine genome-wide profiling O-GlcNAc-modified with perturbations intracellular glycosylation, II-degron, super-resolution microscopy. Genomic shows a non-random distribution across genome, high densities heterochromatin regions as well actively transcribed gene promoters. Large-scale intersection signal at promoters public ChIP-seq datasets identifies overlap specific cofactors. Knockdown Transferase ( Ogt ) that most direct target genes are downregulated, supporting global positive role cellular genes. Rapid degradation results decrease levels encoding factors DNA enzymes. depletion also unexpectedly causes an increase set for machinery. Conclusions This study provides deconvoluted genomic murine human cells. Perturbations or uncover context-specific reciprocal functional interplay between machinery modification.

Language: Английский

Citations

0
Comparative single-cell transcriptomic atlases of drosophilid brains suggest glial evolution during ecological adaptation DOI Creative Commons
Daehan Lee, Michael P. Shahandeh,

Liliane Abuin

et al.

PLoS Biology, Journal Year: 2025, Volume and Issue: 23(4), P. e3003120 - e3003120

Published: April 29, 2025

To explore how brains change upon species evolution, we generated single-cell transcriptomic atlases of the central three closely related but ecologically distinct drosophilids: generalists Drosophila melanogaster and simulans , noni fruit specialist sechellia . The global cellular composition these species’ is well-conserved, predicted a few cell types with different frequencies, notably perineurial glia blood–brain barrier, which validate in vivo Gene expression analysis revealed that evolve at rates patterns, glial populations exhibiting greatest divergence between species. Compared to D. brain, gene patterns are more divergent than —despite their similar phylogenetic distance from —indicating specialization reflected structure function its brain. Expression changes include several metabolic signaling genes, suggestive adaptations novel source nutrition. Additional on genes responsive dietary supplement noni, pointing as sites for both physiological genetic adaptation this fruit. Our represent first comparative datasets “whole” provide comprehensive foundation studying evolvability nervous systems well-defined ecological framework.

Language: Английский

Citations

0
ALT-FISH quantifies alternative lengthening of telomeres activity by imaging of single-stranded repeats DOI Creative Commons
Lukas Frank, Anne Rademacher, Norbert Mücke

et al.

Nucleic Acids Research, Journal Year: 2022, Volume and Issue: 50(11), P. e61 - e61

Published: Feb. 5, 2022

Alternative lengthening of telomeres (ALT) occurs in ∼10% cancer entities. However, little is known about the heterogeneity ALT activity since robust detection assays with high-throughput situ readouts are lacking. Here, we introduce ALT-FISH, a method to quantitate single cells from accumulation single-stranded telomeric DNA and RNA. It involves one-step fluorescent hybridization approach followed by fluorescence microscopy imaging. Our reliably identified cell lines different tumor entities was validated three established models induction suppression. Furthermore, successfully applied ALT-FISH spatially resolve primary tissue sections leiomyosarcoma neuroblastoma tumors. Thus, our assay provides insights into tumors suited for applications, which will facilitate screening ALT-specific drugs.

Language: Английский

Citations

17