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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 <t>DRAQ7-positive</t> 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.
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draq7 dye  (Novus Biologicals)
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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 <t>DRAQ7-positive</t> 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.
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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 <t>DRAQ7-positive</t> 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.
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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 <t>DRAQ7-positive</t> 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.
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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 <t>DRAQ7-positive</t> 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.
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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 <t>DRAQ7-positive</t> 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.
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dna dye draq 7  (Cell Signaling Technology Inc)
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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 <t>DRAQ7-positive</t> 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.
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draq7 drop & go dye d15107  (Thermo Fisher)
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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 <t>DRAQ7-positive</t> 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.
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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.

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