Journal: bioRxiv

Article Title: CPSF3 inhibition blocks pancreatic cancer cell proliferation through disruption of core histone processing

doi: 10.1101/2022.05.09.491230

Figure Lengend Snippet: A, Changes in PDUI score (-0.1 > PDUI > 0.1; P<0.05) denoting 3′-UTR-shortened (red) and lengthened (blue) genes. B, Genes showing lengthening (right) or shortening (left) events (-0.1> PDUI > 0.1; P<0.05) and are differentially expressed (FDR<0.05; fold change >1.5) as color coded. Up=upregulated gene expression, Down=downregulated gene expression. C, Venn diagram showing overlapping genes with significant APA alterations between JTE-607- treated and CPSF3 knockdown cells. D, Heatmap of differentially expressed genes in Panc1 cells treated with JTE-607. Expression is plotted as transformed expression value. Blue triangles denote replication-dependent histone genes. E, Gene set enrichment analysis (GSEA) of RNA-seq data from (D) . F, mRNA expression of H2B and H3 in MiaPaCa2 cells treated with JTE-607. * , P < 0.05, ** , P < 0.01, *** , P < 0.001, Ordinary one-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: Primary antibodies were diluted in 3% BSA in TBST and incubated overnight at 4°C (mouse monoclonal CPSF3 antibody, Abcepta, AT1610a; rabbit polyclonal Histone H3 antibody, Cell Signaling Technology, 9715S; rabbit polyclonal Histone H2B antibody, Cell Signaling Technology, 8135S; mouse monoclonal GAPDH antibody, Proteintech, 60004-1-Ig; rabbit polyclonal FHL1 antibody, Proteintech, 10991-1-AP).

Techniques: Expressing, Transformation Assay, RNA Sequencing Assay

Journal: bioRxiv

Article Title: CPSF3 inhibition blocks pancreatic cancer cell proliferation through disruption of core histone processing

doi: 10.1101/2022.05.09.491230

Figure Lengend Snippet: A, IGV-generated density plots of replication-dependent histones highlighting the differences of 3′- UTR coverage between DMSO (red) and JTE-607 (blue) treated cells. B, Western blot of H2B and H3 protein levels in Panc1 cells treated with 0-10μM JTE-607 for 24 and 48hrs. C, IGV-generated density plots of replication-independent histones highlighting the differences of 3′-UTR coverage between DMSO (red) and JTE-607 (blue) treated cells. D, DSeq2 normalized counts of H2AFZ and H3F3A histone variants in Panc1 cells treated with JTE-607. ** , P < 0.001. E-G, Volcano plots of Spearman’s correlation of CPSF3 and: E, all histone genes; F, replication-dependent histone genes; G, replication-independent histone genes. Each dot represents a histone gene. Blue and red dots denote positive and negative correlation, respectively. (Spearman = -0.15>R>0.15, P<0.05).

Article Snippet: Primary antibodies were diluted in 3% BSA in TBST and incubated overnight at 4°C (mouse monoclonal CPSF3 antibody, Abcepta, AT1610a; rabbit polyclonal Histone H3 antibody, Cell Signaling Technology, 9715S; rabbit polyclonal Histone H2B antibody, Cell Signaling Technology, 8135S; mouse monoclonal GAPDH antibody, Proteintech, 60004-1-Ig; rabbit polyclonal FHL1 antibody, Proteintech, 10991-1-AP).

Techniques: Generated, Western Blot

Journal: Molecular cell

Article Title: Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency

doi: 10.1016/j.molcel.2021.06.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-Histone H2B (V119) , Cell Signaling Technology , Cat# 8135; RRID: AB_10891053.

Techniques: Recombinant, Electron Microscopy, Plasmid Preparation, Imaging, CCK-8 Assay, CRISPR, shRNA, Negative Control, Construct, Software, Transfection, DNA Extraction

Journal: Cell

Article Title: Ubiquitination-Deficient Mutations in Human Piwi Cause Male Infertility by Impairing Histone-to-Protamine Exchange during Spermiogenesis

doi: 10.1016/j.cell.2017.04.034

Figure Lengend Snippet: (A) Western blotting of ub-H2A, ub-H2B, and H4K16ac in late spermatids from dpp 12-week mice with H3K27me3 as a loading control. Quantification of blotting intensity for indicated proteins is shown in parentheses (the one in Miwi+/DB control is set as 100% after normalization with H3K27me3 blotting).

Article Snippet: Rabbit polyclonal anti-H2B , Cell Signaling Technology , Cat#12364.

Techniques: Western Blot

Journal: Cell

Article Title: Ubiquitination-Deficient Mutations in Human Piwi Cause Male Infertility by Impairing Histone-to-Protamine Exchange during Spermiogenesis

doi: 10.1016/j.cell.2017.04.034

Figure Lengend Snippet: (A–C) Immunostaining of endogenous RNF8 (A), (red) or ub-H2B (B), (red) in late spermatids (LS) and H2B in sperm (C), (red) from Miwi+/DB-Cre males transduced by RNF8-N:Cyto IV-EGFP (left) or RNF8-Nmut:Cyto IV-EGFP (right), with nuclei counterstained by DAPI (blue). Left lanes in each staining panel show the representative staining images, and GFP (green) and arrows indicate cells that were successfully transduced with the respective constructs; right lanes show an enlarged view of the representative cells. Right panels: quantification of late spermatids showing visible RNF8 nucleus translocation (A) or ub-H2B staining (B), and sperm without H2B staining (C), top) or with normal morphology (C), bottom) among GFP+ or GFP− cells (n = 100 per group; the average values ± SD of three separate experiments are plotted). Scale bar, 10 μm.

Article Snippet: Rabbit polyclonal anti-H2B , Cell Signaling Technology , Cat#12364.

Techniques: Immunostaining, Staining, Transduction, Construct, Translocation Assay

Journal: Cell

Article Title: Ubiquitination-Deficient Mutations in Human Piwi Cause Male Infertility by Impairing Histone-to-Protamine Exchange during Spermiogenesis

doi: 10.1016/j.cell.2017.04.034

Figure Lengend Snippet: Key Resources Table

Article Snippet: Rabbit polyclonal anti-H2B , Cell Signaling Technology , Cat#12364.

Techniques: Recombinant, In Situ, Sequencing, Software

Journal: Cell

Article Title: Ubiquitination-Deficient Mutations in Human Piwi Cause Male Infertility by Impairing Histone-to-Protamine Exchange during Spermiogenesis

doi: 10.1016/j.cell.2017.04.034

Figure Lengend Snippet: (A) Western blotting of ub-H2A, ub-H2B, and H4K16ac in late spermatids from dpp 12-week mice with H3K27me3 as a loading control. Quantification of blotting intensity for indicated proteins is shown in parentheses (the one in Miwi+/DB control is set as 100% after normalization with H3K27me3 blotting).

Article Snippet: Rabbit polyclonal anti-ub-H2B , Cell Signaling Technology , Cat#5546; RRID:AB_10693452.

Techniques: Western Blot

Journal: Cell

Article Title: Ubiquitination-Deficient Mutations in Human Piwi Cause Male Infertility by Impairing Histone-to-Protamine Exchange during Spermiogenesis

doi: 10.1016/j.cell.2017.04.034

Figure Lengend Snippet: (A–C) Immunostaining of endogenous RNF8 (A), (red) or ub-H2B (B), (red) in late spermatids (LS) and H2B in sperm (C), (red) from Miwi+/DB-Cre males transduced by RNF8-N:Cyto IV-EGFP (left) or RNF8-Nmut:Cyto IV-EGFP (right), with nuclei counterstained by DAPI (blue). Left lanes in each staining panel show the representative staining images, and GFP (green) and arrows indicate cells that were successfully transduced with the respective constructs; right lanes show an enlarged view of the representative cells. Right panels: quantification of late spermatids showing visible RNF8 nucleus translocation (A) or ub-H2B staining (B), and sperm without H2B staining (C), top) or with normal morphology (C), bottom) among GFP+ or GFP− cells (n = 100 per group; the average values ± SD of three separate experiments are plotted). Scale bar, 10 μm.

Article Snippet: Rabbit polyclonal anti-ub-H2B , Cell Signaling Technology , Cat#5546; RRID:AB_10693452.

Techniques: Immunostaining, Staining, Transduction, Construct, Translocation Assay

Journal: Cell

Article Title: Ubiquitination-Deficient Mutations in Human Piwi Cause Male Infertility by Impairing Histone-to-Protamine Exchange during Spermiogenesis

doi: 10.1016/j.cell.2017.04.034

Figure Lengend Snippet: Key Resources Table

Article Snippet: Rabbit polyclonal anti-ub-H2B , Cell Signaling Technology , Cat#5546; RRID:AB_10693452.

Techniques: Recombinant, In Situ, Sequencing, Software