Journal: Scientific Reports

Article Title: FOXK2 Transcription Factor Suppresses ERα-positive Breast Cancer Cell Growth Through Down-Regulating the Stability of ERα via mechanism involving BRCA1/BARD1

doi: 10.1038/srep08796

Figure Lengend Snippet: (a) HEK 293T cells transfected with HA-BARD1 only, or with HA-BARD1 and His-Flag-FOXK2 were subjected to immunoprecipitation with anti-Flag antibody followed by Western blot with anti-HA antibody or vice versa. (b) HEK 293T cells transfected with appropriate plasmids were subjected to immunoprecipitation and Western blot with specific antibodies as indicated or vice versa. (c) MCF-7 cells were subjected to immunoprecipitation with anti-MDM2 antibody followed by Western blot with anti-FOXK2 antibody or vice versa. (d) MCF-7 cells were subjected to immunoprecipitation with anti-BARD1 antibody followed by Western blot with anti-FOXK2 antibody or vice versa. (e) HEK 293T cells transfected with HA-BARD1 and different FOXK2 constructs as indicated were collected and then subjected to immunoprecipitation with anti-HA antibody, followed by Western blot analysis with anti-Flag antibody. (f) MCF-7 cells transfected with EGFP-FOXK2 and HA-BARD1 were stained with rabbit anti-HA antibody and TRITC-conjugated anti-rabbit IgG, and then counterstained with DAPI (blue) for nucleus detection. EGFP-FOXK2 appeared as green signal when visualized by fluorescence microscopy. (g) MCF-7 cells transfected with Flag-ERα and HA-BARD1 were subjected to immunoprecipitation with anti-Flag antibody and re-immunoprecipitation (RE) with anti-HA antibody followed by Western blot with anti-FOXK2 antibody, anti-Flag or anti-HA antibody. (h) HEK 293T cells transfected with appropriate plasmids as indicated, and then treated with 10 μM MG132 for 8 h. Cells were collected and then subjected to immunoprecipitation with anti-Flag antibody followed by Western blot with the indicated antibodies. (i) HEK 293T cells transfected with appropriate plasmids as indicated, and then treated with 10 μM MG132 for 8 h. Cells were collected and then subjected to immunoprecipitation with anti-Flag antibody followed by Western blot with the indicated antibodies. (j) HEK 293T cells transfected with various combinations of different constructs as indicated were treated with 10 μM MG132 for 8 h. The cells were collected and subjected to immunoprecipitation with anti-ERα antibody followed by Western blot analysis with anti-Myc antibody. All experiments were repeated at least three times. The full-length blot of is presented in .

Article Snippet: Rabbit polyclonal anti-BARD1 antibody was obtained from BIOSS (Beijing, China).

Techniques: Transfection, Immunoprecipitation, Western Blot, Construct, Staining, Fluorescence, Microscopy

Journal: Cell discovery

Article Title: The BRCA1/BARD1 complex recognizes pre-ribosomal RNA to facilitate homologous recombination.

doi: 10.1038/s41421-023-00590-8

Figure Lengend Snippet: Fig. 4 Pre-rRNA targets the BRCA1/BARD1 complex to DSBs. a Pre-rRNP exists at IRIF. Cells were treated with 10 Gy of IR. Pre-rRNA was examined by RNA probes. Ribosomal proteins were stained with indicated antibodies. The colocalization with BRCA1 was analyzed (bottom panel). b Foci number per cell and foci colocalization ratio are examined. c The IRIF of the BRCA1/BARD1 complex in the pol Ii-treated cells. HeLa cells were treated with RNA polymerase inhibitors prior to 10 Gy of IR. The IRIF of BRCA1 and BARD1 were stained with indicated antibodies. Foci number per cell is shown (right panel). The cells were also counterstained by DAPI. P values were calculated using Student’s t-test. Scale bars, 10 μm. d A schematic diagram showing that pre-incubation with pre-rRNA blocks the binding of the recombinant BRCA1 BRCT or BARD1 BRCT to IRIF (top left panel). Foci were examined with anti-GST antibody (bottom panel). DSB foci were marked with an anti-γH2AX antibody. Foci number per cell is shown (top right panel). P values were calculated using Student’s t test. n.s. nonsignificant, *P < 0.05, ***P < 0.001. Scale bars, 10 μm. e Enrichment of pre-rRNA at the unsynapsed axis of X and Y chromosomes. Analysis of pre-rRNA and SCP3 distribution is shown (right panel). f Pre-incubation of the BRCTs with pre- rRNA abolishes their localization onto the XY body. Recombinant BRCT proteins were pre-incubated with pre-rRNA. The circled area indicates the XY body. Scale bars, 10 μm.

Article Snippet: Anti-BARD1 antibody was purchased from Novus.

Techniques: Staining, Incubation, Binding Assay, Recombinant

Journal: Journal of Translational Medicine

Article Title: Survival advantage of native and engineered T cells is acquired by mitochondrial transfer from mesenchymal stem cells

doi: 10.1186/s12967-024-05627-4

Figure Lengend Snippet: MitoT increases the expression of anti-apoptotic Bcl-2/BARD1 pathways. A Fold change of differentially expressed (DE) genes contained within the GO terms related to apoptosis, cell death, and/or responses to different stimuli. B qRT-PCR analysis of BARD1 mRNA expression levels in FACS-sorted CD3+ T cells after 24-h post-mitoception with MSC derived-MT (n = 4). C Representative western blots of BARD1 in FACS-sorted CD3+ MitoT pos cells and MitoT neg cells after 24 or 48 h post-mitoception. D Fold change quantification of proteins showed in C (n = 3). E Ingenuity Pathway analysis for Bcl-2 gene network for the DE genes denoted in A . F qRT-PCR of Bcl-2 family gene expression in FACS-sorted CD3+ MitoT pos cells and MitoT neg cells after 24 h post-mitoception (n = 4). For figures B and D : graphs show mean ± SEM and statistical analysis by unpaired t-test. For figure F : graph shows mean ± SEM and statistical analysis by unpaired Mann–Whitney test (*p < 0.05, relative to MitoT neg control). All replicates are biological

Article Snippet: Then membranes were incubated with 1:1000 primary antibodies, rabbit anti-Caspase 3, or mouse anti-β-actin (Cell Signaling Technology #9662 and #3700, respectively), and rabbit anti-BRCA1-associated RING domain protein 1 (BARD1) (Novus Biological #NB100-319) and incubated overnight at 4 °C with shaking.

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Western Blot, Gene Expression, MANN-WHITNEY, Control

Journal: Journal of Biological Chemistry

Article Title: The BRCA1 RING and BRCT Domains Cooperate in Targeting BRCA1 to Ionizing Radiation-induced Nuclear Foci

doi: 10.1074/jbc.m408879200

Figure Lengend Snippet: FIG. 1. Endogenous BRCA1 and ec- topic YFP-BRCA1 form nuclear foci with BARD1 and MDC1. A, endogenous BRCA1 nuclear foci (stained with Ab-4 and Texas Red) co-localize with BARD1 and MDC1 (stained with fluorescein iso- thiocyanate) in T47D cells before and af- ter 15-Gy IR treatment and 4 h of recov- ery. B, YFP-tagged wild-type BRCA1 was transfected into MCF-7 cells and assessed 48 h later for nuclear foci, which also co- localized with BARD1 and MDC1 (stained with Texas Red). Note the redistribution of BRCA1 from few and larger spots in untreated cells to small and more dis- persed foci in IR-treated cells.

Article Snippet: For BARD1 and MDC1 staining, rabbit BARD1 antibody 59L (gift from Professor Richard Baer) and rabbit MDC1 BL578 antibody (Bethyl Laboratories) were used, respectively.

Techniques: Staining, Transfection

Journal: Journal of Biological Chemistry

Article Title: The BRCA1 RING and BRCT Domains Cooperate in Targeting BRCA1 to Ionizing Radiation-induced Nuclear Foci

doi: 10.1074/jbc.m408879200

Figure Lengend Snippet: FIG. 2. Effect of BRCA1 cancer mutations on nuclear focus formation. A, diagram of BRCA1 showing the location of RING and BRCT domains and of published nuclear localization (NLS) and nuclear export (NES) transport signals. YFP-tagged BRCA1 wild-type and mutant proteins were co-transfected with pFLAG-BARD1 into MCF-7 cells and either left untreated or irradiated with 15 Gy of IR with 4 h of recovery. Representative images of nuclei and foci are shown. B, nuclear foci scoring results for the wild-type and mutant BRCA1. Scores were categorized into no focus, 1–10 foci, and 10 foci, and for each category an average percentage of transfected cells (S.E.) was obtained from two separate experiments (100 cells per experiment). Data for wild-type BRCA1 was from four experiments (mean S.D.). White bars represent untreated samples, and black bars represent irradiated samples.

Article Snippet: For BARD1 and MDC1 staining, rabbit BARD1 antibody 59L (gift from Professor Richard Baer) and rabbit MDC1 BL578 antibody (Bethyl Laboratories) were used, respectively.

Techniques: Mutagenesis, Transfection, Irradiation

Journal: Journal of Biological Chemistry

Article Title: The BRCA1 RING and BRCT Domains Cooperate in Targeting BRCA1 to Ionizing Radiation-induced Nuclear Foci

doi: 10.1074/jbc.m408879200

Figure Lengend Snippet: FIG. 7. RING/BARD1-binding fragment inhibits endogenous BRCA1 nuclear focus formation. A, five short YFP-BRCA1 constructs were transfected into T47D cells to assess their effect on endogenous BRCA1 (stained with Ab-4 and Texas Red). Cells were left untreated or treated with 15 Gy of IR and 4 h of recovery. Cell images show representative transfected cells and co-staining for endogenous BRCA1 and cell nuclei (stained with Hoechst). The arrows point to transfected cell nuclei. B, endogenous BRCA1 nuclear foci were counted in cells transfected with the YFP-BRCA1 constructs (cells with no visible BRCA1 not shown in graphs). Cells were scored for 0, 1–10, and 10 foci; white bars and black bars represent untreated and irradiated samples, respectively.

Article Snippet: For BARD1 and MDC1 staining, rabbit BARD1 antibody 59L (gift from Professor Richard Baer) and rabbit MDC1 BL578 antibody (Bethyl Laboratories) were used, respectively.

Techniques: Binding Assay, Construct, Transfection, Staining, Irradiation

Journal: Nucleic Acids Research

Article Title: BRCA1-BARD1 regulates transcription through BRD4 in Xenopus nucleoplasmic extract

doi: 10.1093/nar/gkab111

Figure Lengend Snippet: BRCA1-BARD1 is necessary and sufficient to suppress transcription in NPE. ( A ) Different amounts of mock-depleted (ΔMock) or BRCA1-depleted (ΔBRCA1) NPE was analyzed by western blot with the indicated antibodies. ( B ) pActin was incubated in mock- or BRCA1-depleted extract and RNA was quantified by RT-qPCR over time ( n = 3). ( C ) Purified BRCA1-BARD1 (WT) and BRCA1 I26A -BARD1 (I26A; used in Figure ) were resolved by SDS-PAGE and visualized by silver stain. ( D ) pActin was incubated in mock-depleted extract, BRCA1-depleted extract, or BRCA1-depleted extract supplemented with recombinant 200 nM BRCA1-BARD1. After 120 min, RNA was quantified by RT-qPCR ( n = 2) ( E ) pActin was incubated in NPE supplemented with increasing amounts of BRCA1-BARD1. After 120 min, RNA was quantified by RT-qPCR ( n = 3).

Article Snippet: Xenopus BRCA1 and BARD1 antibodies were generated by New England Peptide.

Techniques: Western Blot, Incubation, Quantitative RT-PCR, Purification, SDS Page, Silver Staining, Recombinant

Journal: Nucleic Acids Research

Article Title: BRCA1-BARD1 regulates transcription through BRD4 in Xenopus nucleoplasmic extract

doi: 10.1093/nar/gkab111

Figure Lengend Snippet: BRCA1 regulates transcription initiation through a histone intermediate. ( A ) 2.5 ng/μl pActin was incubated in mock- or BRCA1-depleted extract with increasing amounts of carrier plasmid. After 120 min, RNA was quantified by RT-qPCR ( n = 2). For comparison, values were normalized to ΔMock reactions at each concentration. ( B ) pActin was incubated in mock- or BRCA1-depleted extract. Samples were withdrawn after 60 min and analyzed by ChIP with histone H3 antibodies ( n = 3). ( C ) pActin was incubated in extract supplemented with buffer or 200 nM BRCA1-BARD1. Samples were withdrawn after 60 min and analyzed by ChIP with histone H3 antibodies ( n = 3). ( D , E ) TBP- and RNAPII-ChIPs were performed as in (B) ( n = 3). ( F – H ) Sperm chromatin was incubated in mock- or BRCA1-depleted extract. At the indicated time points, samples were visualized by phase contrast light microscopy (F). Chromatin volume (G) and density (H) were calculated at 150 min ( n ≥ 12).

Article Snippet: Xenopus BRCA1 and BARD1 antibodies were generated by New England Peptide.

Techniques: Incubation, Plasmid Preparation, Quantitative RT-PCR, Comparison, Concentration Assay, Light Microscopy

Journal: Nucleic Acids Research

Article Title: BRCA1-BARD1 regulates transcription through BRD4 in Xenopus nucleoplasmic extract

doi: 10.1093/nar/gkab111

Figure Lengend Snippet: Ubiquitination is dispensable for BRCA1-mediated transcription suppression. ( A ) pActin was incubated in mock- or BRCA1-depleted extract. Samples were withdrawn after 60 min and analyzed by ChIP with histone H2A-Ub or H3 antibodies. Recovery of H2A-Ub over H3 is graphed ( n = 2). ( B ) pActin was incubated in extract supplemented with buffer or 200 nM BRCA1-BARD1. Samples were analyzed by ChIP as in (A) ( n = 2). ( C , D ) pActin was incubated in extract pre-treated with buffer or UbVS and then supplemented with buffer or 200 nM BRCA1-BARD1. Samples were withdrawn after 60 min and analyzed by western blot (C), or 120 min and RNA was quantified by RT-qPCR (D) ( n = 2). ( E ) pActin was incubated in NPE supplemented with increasing amounts of BRCA1 I26A -BARD1. After 120 min, RNA was quantified by RT-qPCR ( n = 3). Results from Figure are also shown for comparison.

Article Snippet: Xenopus BRCA1 and BARD1 antibodies were generated by New England Peptide.

Techniques: Ubiquitin Proteomics, Incubation, Western Blot, Quantitative RT-PCR, Comparison

Journal: Nucleic Acids Research

Article Title: BRCA1-BARD1 regulates transcription through BRD4 in Xenopus nucleoplasmic extract

doi: 10.1093/nar/gkab111

Figure Lengend Snippet: BRCA1-BARD1 regulates DNA-binding of multiple transcription regulators. ( A ) pActin was incubated in extract that was mock-depleted, BRCA1-depleted, or supplemented with 200 nM BRCA1-BARD1. DNA-bound proteins were isolated by plasmid pull-down and analyzed by mass spectrometry. Results were normalized to ΔMock samples and ranked by enrichment of ΔBRCA1 over +BRCA1-BARD1 samples. Representative complex members are labeled. An extended graph showing distribution of ranked proteins is shown in . ( B ) Schematic showing the relationship between BRCA1, BRD4, and complexes identified in (A) .

Article Snippet: Xenopus BRCA1 and BARD1 antibodies were generated by New England Peptide.

Techniques: Binding Assay, Incubation, Isolation, Plasmid Preparation, Mass Spectrometry, Labeling

Journal: Nucleic Acids Research

Article Title: BRCA1-BARD1 regulates transcription through BRD4 in Xenopus nucleoplasmic extract

doi: 10.1093/nar/gkab111

Figure Lengend Snippet: BRCA1-BARD1 suppresses H4K8 acetylation and BRD4 binding. ( A ) pActin was incubated in mock- or BRCA1-depleted extract. At the indicated time points, DNA-bound proteins were isolated by plasmid pull-down and analyzed by western blot. Total recovery of plasmid DNA from mock- and BRCA1-depleted extracts is shown in . ( B ) pActin was incubated in extract supplemented with buffer or 200 nM BRCA1-BARD1 and DNA-bound proteins were analyzed as in (A). ( C ) pActin was incubated in extract supplemented with buffer or JQ1 (BETi). At the indicated time points, DNA-bound proteins were analyzed as in (A). ( D ) pActin was incubated in extract with increasing amounts of JQ1. After 120 min, RNA was quantified by RT-qPCR ( n = 3). ( E ) pActin was incubated in NPE supplemented with buffer or JQ1. At the indicated time points, RNA was quantified by RT-qPCR. ( F ) pActin was incubated in mock- or BRCA1-depleted extract. Samples were withdrawn after 20 min and analyzed by ChIP with the indicated antibodies ( n = 2). ( G ) pActin was incubated in extract supplemented with buffer or JQ1. Samples were withdrawn after 20 min and analyzed by ChIP with the indicated antibodies ( n = 2).

Article Snippet: Xenopus BRCA1 and BARD1 antibodies were generated by New England Peptide.

Techniques: Binding Assay, Incubation, Isolation, Plasmid Preparation, Western Blot, Quantitative RT-PCR