b23319 draq7 cell signaling  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Paired CRISPR screens identify mitochondrial metabolism and UBE2H as aneuploid-specific dependencies in human cancer cell lines

doi: 10.64898/2026.04.26.720636

Figure Lengend Snippet: Validation and mechanistic analysis of UBE2H A-D) Competition assay results showing fold dropout of sgRNAs targeting negative controls (ROSA26, AAVS1), positive controls (CDK9, CDC7), and UBE2H in A) SNU1 near-tetraploid control cells, B) SNU1 aneuploid subclone 114 (trisomy 9, 19), C) SNU1 aneuploid subclone 12 (trisomy 5, 10,17; pentasomy 2), D) SNU1 aneuploid subclone 24 (trisomy 1,8,10,14; DiY; pentasomy 5). Percent dropout is normalized relative to positive and negative controls. E) Colony formation following AZ3146 treatment (250 nM) relative to ethanol vehicle control in HCT116 wild-type cells, three UBE2H knockout clones, and UBE2H cDNA rescue clones. P-values from two-sided t-test relative to wild-type are shown. One sided t-test for overexpression clones rescue relative to knockout. F) Change in percentage of Annexin V and/or DRAQ7-positive cells following 96 h treatment with AZ3146 (1 µM), relative to ethanol-treated controls in HCT116 wild-type and UBE2H-KO clones. Two-sided t-tests. G) Diagram of sample preparation for Mass Spectrometry analysis. H-J) g:Profiler gene group enrichment analysis of mass spectrometry data. Protein abundance downregulated in HCT116 in H) AZ3146 treated versus wild-type, I) UBE2H-KO versus wild-type and J) AZ3146 treated UBE2H-KO versus AZ3146 treated wild-type cells. K) UBE2H RNA expression correlated to aneuploidy score in TCGA human tumor data. Pearson correlation and corresponding p-value. L) UBE2H RNA expression stratified by chromosome arm 7q copy number status; with 7q arm loss (-1), neutral (0) and gain (1) in TCGA data. Two-sided t-test p-values are shown. M) UBE2H RNA expression correlated to aneuploidy score for samples with a neutral copy of chromosome arm 7q in TCGA data. N) UBE2H RNA expression and aneuploidy score Pearson correlation coefficient and -log₂(p-value) per tumor types (TCGA). All significant correlations labeled with tumor abbreviation.

Article Snippet: Cells and media were harvested, resuspended in 100 μl annexin buffer and stained with AnnexinV FITC conjugate (Fisher Scientific, A13199) and then with 1 μl of DRAQ7 dead cell dye (Fisher Scientific, D15106).

Techniques: Biomarker Discovery, Competitive Binding Assay, Control, Knock-Out, Clone Assay, Over Expression, Sample Prep, Mass Spectrometry, Quantitative Proteomics, RNA Expression, Labeling

Journal: eLife

Article Title: Control of innate olfactory valence by segregated cortical amygdala circuits

doi: 10.7554/eLife.104677

Figure Lengend Snippet: Related to . ( A ) Representative images of tissue microdissection sites from aplCoA and pplCoA following extraction and DAPI staining (blue). Scale bars, 500 µm. ( B ) Location of all tissue sample sites used for snRNA-seq, color coded by plCoA zone (n=3 pools per zone, 4–11 sections per pool). Scale bars, 500 µm. ( C ) Validation of nuclear enrichment after FANS. Ethidium homodimer-1 (EthD-1, red) labels nuclei on a hemocytometer after sorting, with an absence of non-nuclear, EthD-1-negative debris. Scale bar, 100 µm. ( D ) Common gating strategy for FANS sorts for snRNA-seq in a representative sample. Far left, morphology gate on forward and side scatter area excludes likely debris. Middle left, forward scatter gate excludes nuclear doublets with high forward scatter width. Middle right, side scatter gate excludes nuclear multiplets with high side scatter width. Far right, stoichiometric DRAQ7+ fluorescence allows enrichment of single nuclei and exclusion of debris and multiplets. ( E ) Absolute number and proportion of snRNA-seq nuclei passing quality control filters from each replicate in each plCoA zone (n=27,726 in aplCoA, 19,406 in pplCoA, 3 libraries/batches each). ( F ) Violin plot of UMIs detected per snRNA-seq nucleus for each replicate, filtered at the median per library +five times the median absolute deviation within each library (median 6081 UMIs/nucleus). ( G ) Violin plot of genes detected per nucleus from each replicate, filtered at a minimum of 1000 features per nucleus (median 2547 genes/nucleus). ( H ) Percent mitochondrial gene UMIs per snRNA-seq nucleus, filtered at median +five times the median absolute deviation per library (median 0.02% mitochondrial UMIs/nucleus). ( I ) Percent ribosomal gene UMIs per snRNA-seq nucleus, filtered at median +five times the median absolute deviation per library (median 0.17% ribosomal UMIs/nucleus). ( J ) Principal component analysis of pseudobulk snRNA-seq samples created from each batch, colored based on their combination of zone and batch identity. ( K ) Evaluation of transcriptomic homology between batches, where the distance matrix is based on Spearman correlation between median expression of highly variable features for the whole dataset, and the dendrogram was created via hierarchical clustering of batches on this correlation matrix. ( L ) UMAP of all snRNA-seq nuclei colored by both target region and batch identity. ( M ) Relative proportion of nuclei of each type for all snRNA-seq batches. Brain diagrams were reproduced from .

Article Snippet: Nuclei were finally resuspended in nuclear flow buffer containing 3 μm DRAQ7 (Cell Signaling Technology) and again filtered through a 40 μm Flowmi cell strainer into a 5 ml round-bottom polystyrene tube.

Techniques: Laser Capture Microdissection, Extraction, Staining, Biomarker Discovery, Fluorescence, Control, Expressing