Journal: Science Advances

Article Title: Identification and characterization of BEND2 as a key regulator of meiosis during mouse spermatogenesis

doi: 10.1126/sciadv.abn1606

Figure Lengend Snippet: ( A ) Schematic diagram of the primary structures of the Bend2 gene and BEND2 protein. Top diagram shows the location of Scml2 and Bend2 genes in the X chromosome and the structure of the Bend2 gene; blue and red arrows indicate Scml2 and Bend2 genes; blue and white rectangles indicate protein-coded exons and UTR regions, respectively. Bottom diagram represents the BEND2 protein, with orange boxes indicating the BEN domain. ( B ) RT-PCR detection of Bend2 expression in multiple mouse organs. ( C ) Schematic representation of the locus of the 3xFLAG tag KI; the tag sequence was inserted immediately behind the first codon of BEND2. ( D ) Western blot analyses of BEND2 expression in multiple mouse organs using mmAb-FLAG. ( E ) Immunohistochemical (IHC) staining of FLAG-BEND2 in testicular sections of various seminiferous stages using mmAb-FLAG; red and blue arrows indicate BEND2 strongly and weakly expressing cells, respectively; black arrows indicate no BEND2 expression. SG-B, type B spermatogonia; plpSC, pre-leptotene spermatocytes; lepSC, leptotene spermatocytes; zygSC, zygotene spermatocytes; pacSC, pachytene spermatocytes; dipSC, diplotene spermatocytes; rST, round spermatids; eST, elongated spermatids. Scale bar, 20 μm. ( F ) Schematic summary of FLAG-BEND2 expression in male germ cell types and seminiferous stages. ( G ) Immunofluorescence staining of FLAG-BEND2 shown at higher magnification of BEND2 signals in lepSCs. Scale bars, 10 μm. aa, amino acids.

Article Snippet: An antibody to mouse BEND2 (rpAb-B2-1) was produced by ABclonal Technology (Wuhan, China) and generated by immunization of rabbits with the peptide sequence DVRESVKRERVDFEHTPDANPEGSDNASIN-C (amino acids 585 to 614).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Sequencing, Western Blot, Immunohistochemical staining, Immunohistochemistry, Immunofluorescence, Staining

Journal: Science Advances

Article Title: Identification and characterization of BEND2 as a key regulator of meiosis during mouse spermatogenesis

doi: 10.1126/sciadv.abn1606

Figure Lengend Snippet: ( A ) Schematic illustration of the deletion of two exons of the Bend2 gene to generate Bend2 −4k/Y mice; p1 and p2 indicate that two primers were used in mouse genetic identification. Right diagram shows BEND2 protein structure in WT and KO mice. ( B ) Identification of genotype with p1 (WT allele) and p2 (mutant allele) primer pairs. ( C ) Western blot confirmation of the elimination of BEND2 protein in Bend2 −4k/Y mice using rpAb-B2-2. ( D ) Note the size reduction in 8-week-old Bend2 −4k/Y testes. ( E ) Quantitative comparison of testis/body ratios between Bend2 +/Y and Bend2 −4k/Y mice (*** P < 0.001, Student’s t test). ( F ) Comparison of litter size in Bend2 +/Y and Bend2 −4k/Y mice (**** P < 0.0001, Student’s t test). ( G ) H&E staining of testicular sections in Bend2 +/Y and Bend2 −4k/Y mice. zygSC-like, zygotene-like spermatocytes; apo, apoptotic cells. Scale bars, 20 μm. ( H ) H&E staining of epididymal sections of Bend2 +/Y and Bend2 −4k/Y mice. Scale bars, 20 μm. ( I ) TUNEL staining of testicular sections in Bend2 +/Y and Bend2 −4k/Y mice; green signals indicate apoptotic cells. Scale bars, 50 μm. ( J ) Quantitative comparison of TUNEL staining shows that both TUNEL-positive cells and tubules were increased in Bend2 −4k/Y mice. ( K and L ) Immunofluorescence staining indicates that the number of PLZF-positive cells (green) was the same between Bend2 +/Y and Bend2 −4k/Y mice. Scale bars, 20 μm. ( M and N ) Quantitative comparison of WT1 immunofluorescence staining of testicular sections in Bend2 +/Y and Bend2 −4k/Y mice. Scale bars, 20 μm. ns, not significant.

Article Snippet: An antibody to mouse BEND2 (rpAb-B2-1) was produced by ABclonal Technology (Wuhan, China) and generated by immunization of rabbits with the peptide sequence DVRESVKRERVDFEHTPDANPEGSDNASIN-C (amino acids 585 to 614).

Techniques: Mutagenesis, Western Blot, Comparison, Staining, TUNEL Assay, Immunofluorescence

Journal: Science Advances

Article Title: Identification and characterization of BEND2 as a key regulator of meiosis during mouse spermatogenesis

doi: 10.1126/sciadv.abn1606

Figure Lengend Snippet: ( A ) LC-MS/MS analysis of enriched protein from co-IP. Protein identification was performed in the presence of wash buffer containing 300 or 500 mM NaCl; WT mice served as background controls ( n = 3 independent biological replicates, with each replicate containing two d-15 mice). ( B ) Co-IP of BEND2 with ZMYM2, LSD1, CHD4, HDAC1, ADNP, and HP1γ in testis from WT and Bend2 FLAG/Y mice. The asterisk-labeled band is the heavy chain of mouse IgG. ( C ) Co-IP Western blot analysis used to confirm the interaction of CHD4 and BEND2 or HDAC1 in the testis. ( D ) Co-IP Western blot analysis to confirm the interaction of CHD4 and HP1γ or ADNP in the testis. ( E ) Each dot represents the number of bright foci per spermatocyte in WT and KO mice (**** P < 0.0001, obtained with a two-tailed, unpaired t test). ( F ) Network of BEND2-interacting proteins.

Article Snippet: An antibody to mouse BEND2 (rpAb-B2-1) was produced by ABclonal Technology (Wuhan, China) and generated by immunization of rabbits with the peptide sequence DVRESVKRERVDFEHTPDANPEGSDNASIN-C (amino acids 585 to 614).

Techniques: Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, Labeling, Western Blot, Two Tailed Test

Journal: Science Advances

Article Title: Identification and characterization of BEND2 as a key regulator of meiosis during mouse spermatogenesis

doi: 10.1126/sciadv.abn1606

Figure Lengend Snippet: ( A ) Heatmap of BEND2 ChIP-seq enrichment across all peaks ( n = 16,477) in the mouse genome. Each row represents a 6-kb window centered on BEND2 peak midpoints, sorted by the BEND2 ChIP signal. Input signals at the same position are shown on the right (average peak intensity of n = 6 biological replicates). ( B ) BEND2-binding sites were classified by their genomic locations and repeat types as indicated. ( C ) Top BEND2 DNA binding motif predicted by HOMER (left); sequences with the top motif reanalyzed by HOMER (right). ( D ) Heatmap of chromatin states produced by ChromHMM based on 10 histone modifications (left); heatmap showing different enrichment for indicated annotations for each state (right).

Article Snippet: An antibody to mouse BEND2 (rpAb-B2-1) was produced by ABclonal Technology (Wuhan, China) and generated by immunization of rabbits with the peptide sequence DVRESVKRERVDFEHTPDANPEGSDNASIN-C (amino acids 585 to 614).

Techniques: ChIP-sequencing, Binding Assay, Produced

Journal: Science Advances

Article Title: Identification and characterization of BEND2 as a key regulator of meiosis during mouse spermatogenesis

doi: 10.1126/sciadv.abn1606

Figure Lengend Snippet: ( A ) The number of up- and down-regulated genes in lep/zygSCs of adult KO mice. The DEGs listed in the tables include both annotated genes with regular gene symbols and unannotated genes with only gene IDs, and only annotated genes were used to perform comparisons between different sets; the numbers of DEGs in the tables are therefore different from those indicated by the Venn diagrams. ( B ) Volcano plot of transcript levels between cells from adult WT and KO mice using zygSCs. The DEGs are highlighted in red (up-regulated in KO mice) and blue (down-regulated in KO mice). ( C ) Representative Gene Ontology (GO) terms of the biological process categories enriched in DEGs of zygSCs. ( D ) Average distribution of ATAC-seq signal around the TSS of three clusters of genes. ( E ) Average distribution of H3K4me3 and H3K27me3 around the TSS of three clusters of genes. ( F ) Correlation analysis between the three clusters of genes by ATAC-seq and DEGs. ( G ) Validation of DNA binding motif of BEND2 using dual-luciferase assay. * P < 0.05. ( H ) Browser view showing BEND2 ChIP-seq, ATAC-seq, and H3K4me3 ChIP-seq signals of target genes (left). Different types of peaks were first related to specific genes, and we noted gene sets associated with the different peaks as being of interest; genes at the intersection of all the sets were then selected to construct the track views. Track views of several genes involved in spermatogonial differentiation and meiosis were ultimately selected to represent the different types of peak localizations. Dual-luciferase assay showing the repression of BEND2 target genes ( n = 3, ** P < 0.01; right).

Article Snippet: An antibody to mouse BEND2 (rpAb-B2-1) was produced by ABclonal Technology (Wuhan, China) and generated by immunization of rabbits with the peptide sequence DVRESVKRERVDFEHTPDANPEGSDNASIN-C (amino acids 585 to 614).

Techniques: Biomarker Discovery, Binding Assay, Luciferase, ChIP-sequencing, Construct

Journal: Medicina

Article Title: Secretory Carcinoma of the Breast with Apocrine Differentiation—A Peculiar Entity

doi: 10.3390/medicina60060924

Figure Lengend Snippet: ( A ) Ob20×: S100 is diffusely positive in the secretory component (green circle) and negative in the apocrine component. ( B ) Ob20×: Polyclonal CEA is diffusely positive in the secretory component (green diamond) and negative in the apocrine component. ( C ) Ob20×: CK5/6 is diffusely positive in the secretory component (green star) and negative in the apocrine component.

Article Snippet: The immunohistochemical panel included the following: ER, EP1 clone, Ready to Use (RTU), Dako PR, PgR636 clone, RTU, Dako AR, Ar441 clone, Concentrated, Dilution 1:50, Dako Ki67, MIB-1 clone, RTU, Dako Her2, c-erbB-2 oncoprotein, Concentrated, Dilution 1:200, Dako S-100, 4C4.9, Concentrated, Dilution 1:150, Zeta Gata-3, L50-823, Concentrated, Dilution 1:100, Zeta CEA, Polyclonal RPab, Concentrated, Dilution 1:250, Biosb CK 5/6, D5/16 B4 clone RTU, Dako.

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: The Arctic AβPP mutation leads to Alzheimer’s disease pathology with highly variable topographic deposition of differentially truncated Aβ

doi: 10.1186/2051-5960-1-60

Figure Lengend Snippet: The list of antibodies used in immunohistochemistry and immunoprecipitation experiments

Article Snippet: rpAb 11pE , Aβ11pE , Synaptic Systems.

Techniques: Immunohistochemistry, Immunoprecipitation

Journal: Acta Neuropathologica Communications

Article Title: The Arctic AβPP mutation leads to Alzheimer’s disease pathology with highly variable topographic deposition of differentially truncated Aβ

doi: 10.1186/2051-5960-1-60

Figure Lengend Snippet: Aβ immunostainings of Sw2 patient’s frontal and temporal cortex and mass spectra of Aβ peptides in Sw2 patient’s temporal cortex. a : Confocal micrograph from Sw2 patient’s temporal cortex shows the targetoid pattern of the plaque: the corona contains Aβ x-42 and the centre Aβ 1-5 species. b : The central accentuation of abAβ arc (specific for the Arctic mutation) immunopositivity suggests that much of the mutated Aβ has preserved N-termini (Sw2, frontal cortex; see Figure g and h). c and d : Pyroglutamate specific abAβ 3pE and abAβ 11pE antibodies demonstrate that much of the Aβ is truncated and glutamates 3 and 11 are cyclised into pyroglutamate and this modification occurs in parallel at both sites. Note the strong positivity in pial arteries (Sw2, temporal cortex, consecutive sections). e : Average mass spectra of Aβ peptides extracted from Sw2 patient’s temporal cortex with formic acid and immunoprecipitated with abAβ 17–24 and abAβ Arc (conformation specific epitope 17-24arc), which precipitate both wild type Aβ and Arctic Aβ (see inserted cartoon). Three peaks corresponding to arcAβ species (with the Arctic mutation) and recognized by Aβ pE specific antibodies (values observed in two independent MALDI-TOF experiments m/z =3061.5 and m/z =3062.8 Da → Aβ11pE-40arc; m/z =3246.2 and m/z =3246.0 Da → Aβ11pE-42arc; m/z =4057.0 Da → Aβ3pE-40arc) as well as two peaks corresponding to arcAβ with full length N-termini ( m/z =4258.7 and m/z= 4259.1 Da → 1-40arc; m/z =4443.5 and m/z=4443.0 Da → 1-42arc) are labelled. Additional values with relative intensities of the peaks, see Additional file : Table S1. ( bar in a 50 μm; bar in b 200 μm for b - d ).

Article Snippet: rpAb 11pE , Aβ11pE , Synaptic Systems.

Techniques: Mutagenesis, Modification, Immunoprecipitation

Journal: Acta Neuropathologica Communications

Article Title: The Arctic AβPP mutation leads to Alzheimer’s disease pathology with highly variable topographic deposition of differentially truncated Aβ

doi: 10.1186/2051-5960-1-60

Figure Lengend Snippet: Immunostainings of Sw2 patient’s cerebellum (a, c, d resp. e, f, h, i represent semiconsecutive sections). a - c : C-terminal and mid-domain antibodies abAβ x-42 , abAβ x-40 and abAβ 17–24 disclose similar pattern. There is abundant deposition in the Purkinje cell layer, from where Aβ deposits extend as streaks towards the surface often loosely following penetrating arteries with distinct CAA. d - i : The more N-terminal (beyond aa 17) abAβ 8–17 (d) ; abAβ 5–10 (e) ; and abAβ 1–5 (f) , as well as the specific abAβ arc (g) render markedly weaker staining of the parenchymal deposits, which is also weaker than with the pyroglutamate specific abAβ 3pE (h) and abAβ 11pE (i) . With all Aβ antibodies blood vessels stain approximately as strongly. ( bar in a 500 μm for all panels).

Article Snippet: rpAb 11pE , Aβ11pE , Synaptic Systems.

Techniques: Staining