Journal: Leukemia

Article Title: The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1 -mutated chronic lymphocytic leukemia

doi: 10.1038/s41375-024-02379-4

Figure Lengend Snippet: A ASE categories, including skipped exon (SE), alternative 3′ splice site usage (A3SS), alternative 5′ splice site usage (A5SS), mutually exclusive exon usage (MXE), and intron retention (RI). B Bar plot displaying numbers of all identified ASEs in the comparison of 18 SF3B1 MUT and 17 SF3B1 WT subset #2 cases across different ASE categories. 163,097 ASEs were detected and involved transcripts of 10,437 genes. C Volcano plot depicting all identified ASEs in the comparison of SF3B1 MUT and SF3B1 WT subset #2 cases. Red dots indicate ASEs that are considered significant (|ΔPSI | ≥ 20% and FDR ≤ 0.01; 80 ASEs). D Bar plot displaying numbers of significant ASEs in the comparison of SF3B1 MUT and SF3B1 WT subset #2 cases across different ASE categories. 80 ASEs were considered significant and involved transcripts of 62 genes. E Heatmap illustrating the individual PSI values for the 80 significant ASEs detected in the comparison of SF3B1 MUT and SF3B1 WT subset #2 cases. 33 ASEs occurred within the same gene at least twice. ASEs are displayed based on the ASE category. The bar plot to the right of the heatmap shows ΔPSI values. The gene affected by each ASE and the corresponding unique ASE ID are listed. Genes that encode ncBAF complex-interacting proteins are depicted in color. For multiple ASEs per gene, only the top ASE is colored, while the others are marked with black asterisks. Additional details of these ASEs are provided in Supplementary Table . F Model of the ncBAF chromatin remodeling complex and previously reported ncBAF complex interactors. Alternatively spliced transcripts identified in SF3B1 MUT subset #2 CLL that encode ncBAF complex-related proteins are depicted in color. G Scattered box plot showing the PSI value distribution, ΔPSI values, and P values (Wilcoxon rank-sum test) for significant ncBAF complex-related ASEs identified in the comparison of SF3B1 MUT and SF3B1 WT subset #2 cases. The specific ASEs for ZEB1 , BRD9 , PLSCR1 , TENT4B , CXXC1 , DCAF16 , UBP1 , DLST , and SERBP1 have the unique ASE IDs ZEB1_SE_27141, BRD9_SE_53886, PLSCR1_SE_92684, PAPD5_SE_94061, CXXC1_SE_102397, DCAF16_SE_4627, UBP1_SE_85897, DLST_A3SS_2409, and SERBP1_A3SS_1321, respectively, in Supplementary Table . The boxes represent the median and the interquartile range (IQR), while the whiskers extend to 1.5 times the IQR from the first and third quartiles. ASE: alternative splicing event; WT: wildtype; MUT: mutated; FDR: false discovery rate; PSI: percent spliced in; VAF: variant allele frequency.

Article Snippet: Cell lines and primary CLL cells were treated with BRD9 inhibitors/degraders, I-BRD9 (SML1534; Sigma-Aldrich), PROTAC BRD9 Degrader-1 (HY-103632; MedChemExpress, New Jersey, NJ, USA), and dBRD9 Hydrochloride (SML2911; Sigma-Aldrich) [ , ].

Techniques: Comparison, Alternative Splicing, Variant Assay

Journal: Leukemia

Article Title: The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1 -mutated chronic lymphocytic leukemia

doi: 10.1038/s41375-024-02379-4

Figure Lengend Snippet: Sashimi plots illustrating the identified ASEs and alternative splicing patterns in four genes that encode ncBAF complex-related proteins in SF3B1 WT versus SF3B1 MUT CLL; ZEB1 and BRD9 exhibit the top two ASEs in the SE category, while DLST and SERBP1 showcase the top two ASEs in the A3SS category. Sashimi plots for other ncBAF complex-related genes, PLSCR1 , TENT4B , CXXC1 , DCAF16 , and UBP1 , are presented in Supplementary Fig. . For each gene, the top two sashimi plots within the gray box illustrate the predicted splice variants in SF3B1 WT versus SF3B1 MUT CLL. The colored arc highlights the primary ASE, while lighter arcs represent additional ASEs if present. The gene map indicates the relative location and order of the detected exons in relation to the sequencing results. For each corresponding gene, the lower two sashimi plots show the coverage and splice junction count data from the aligned long-read RNA-seq data from an SF3B1 WT case (RS24) and an SF3B1 MUT case (RS55), both belonging to subset #2 CLL. The direction of the genes is arranged from left to right. WT: wildtype; MUT: mutated; PSI: percent spliced in.

Article Snippet: Cell lines and primary CLL cells were treated with BRD9 inhibitors/degraders, I-BRD9 (SML1534; Sigma-Aldrich), PROTAC BRD9 Degrader-1 (HY-103632; MedChemExpress, New Jersey, NJ, USA), and dBRD9 Hydrochloride (SML2911; Sigma-Aldrich) [ , ].

Techniques: Alternative Splicing, Sequencing, RNA Sequencing

Journal: Leukemia

Article Title: The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1 -mutated chronic lymphocytic leukemia

doi: 10.1038/s41375-024-02379-4

Figure Lengend Snippet: A Scheme illustrating lentiviral constructs for SF3B1 WT and SF3B1 K700E overexpression under the control of a DOX-inducible promoter. B Bar plots depicting expression levels of endogenous and exogenous SF3B1 upon overexpression of SF3B1 WT and SF3B1 K700E in MEC1 and PCL12 cell lines. The bar plots display the mean values of triplicates with error bars representing the standard deviation. C Scheme illustrating the identified ASE in BRD9 with the regular and alternative splicing pattern. The blue and red boxes illustrate constant and alternative exons, respectively. D Increased expression of the alternative BRD9 transcript upon overexpression of SF3B1 K700E as compared to SF3B1 WT in MEC1 and PCL12 cell lines. The checkerboard scheme at the top of the panel illustrates the experimental conditions. The bar plots display the expression levels of BRD9 transcripts in general (transcripts containing exon 14), the regular BRD9 transcript (transcripts containing exon 14 followed by exon 16), and the alternative BRD9 transcript (transcripts containing exon 14 followed by exon 15) as determined by qPCR. Results are normalized on GAPDH levels and expressed as fold difference between DOX+ and DOX− conditions with corresponding P values (Student’s t -test). The bar plots display the mean values of experimental triplicates, with error bars representing the standard deviation. E Agarose gel electrophoresis images of PCR products spanning from exon 14 to 16 and allowing for exon 15 inclusion show a higher abundance of alternative BRD9 transcript in SF3B1 K700E DOX+. For a complete gel image see Supplementary Fig. . F Assessment of BRD9 splice variants in SF3B1 WT and SF3B1 MUT CLL, AML, and UVM cell lines. MEC1, PGA1, and HG3 are SF3B1 WT CLL cell lines, CII an SF3B1 K666E CLL cell line, HNT34 an SF3B1 K700E AML cell line, and MEL202 an SF3B1 R625G UVM cell line. The bar plots display the expression levels of BRD9 transcripts in general, the regular BRD9 transcript, and the alternative BRD9 transcript as determined by qPCR. Results for BRD9 transcripts in general are normalized on GAPDH levels, whereas regular and alternative BRD9 transcript levels are normalized on levels of BRD9 transcripts in general and expressed as fold difference between MEC1 and other cell lines with corresponding P values (Student’s t -test). The bar plots display the mean values of experimental triplicates, with error bars representing the standard deviation. G Agarose gel electrophoresis image of PCR products spanning from exon 14 to 16 and allowing for exon 15 inclusion shows a higher abundance of alternative BRD9 transcript in SF3B1 MUT cell lines. For a complete gel image see Supplementary Fig. . H Sashimi plots from the SF3B1 WT CLL cell line HG3 and the SF3B1 MUT AML and UVM cell lines, HNT34 and MEL202, respectively, illustrating the alternatively spliced exon 15 in BRD9 . The plots for HG3 are based on direct long-read RNA-seq, and HNT34 (SRR8616208) and MEL202 (SRR12354765) on short-read RNA-seq. I BRD9 splice isoform expression in SF3B1 WT and SF3B1 MUT CLL cell lines determined by isoform-specific antibodies. An antibody specific to the C-terminus of the regular BRD9 isoform detected a single band in both SF3B1 WT and SF3B1 MUT cell lines. Conversely, an antibody targeting a constant epitope within the central part of BRD9, detected two bands in the SF3B1 MUT cell line CII and one band in the SF3B1 WT cell lines MEC1 and PGA1, corresponding to the regular higher molecular weight BRD9 isoform and the alternative lower molecular weight BRD9 isoform. For complete blot images see Supplementary Fig. . WT: wildtype; MUT: mutated; DOX: doxycycline.

Article Snippet: Cell lines and primary CLL cells were treated with BRD9 inhibitors/degraders, I-BRD9 (SML1534; Sigma-Aldrich), PROTAC BRD9 Degrader-1 (HY-103632; MedChemExpress, New Jersey, NJ, USA), and dBRD9 Hydrochloride (SML2911; Sigma-Aldrich) [ , ].

Techniques: Construct, Over Expression, Control, Expressing, Standard Deviation, Alternative Splicing, Agarose Gel Electrophoresis, RNA Sequencing, Molecular Weight

Journal: Leukemia

Article Title: The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1 -mutated chronic lymphocytic leukemia

doi: 10.1038/s41375-024-02379-4

Figure Lengend Snippet: A Scheme illustrating BRD9 splice variants and predicted protein isoforms according to NCBI RefSeq (Annotation release GCF_000001405.40-RS_2023_03). The zoom-in view highlights the predicted protein sequence stemming from exon 15 inclusion in BRD9 . Upon translation of exon 15, a stop codon emerges near the end of the exon, resulting in a shorter splice isoform with an alternative C-terminus. B Scheme depicting lentiviral constructs for overexpression of the regular and alternative BRD9 splice variants. The corresponding DNA and protein sequences are available in Supplementary Information Appendix 1. C Heatmap illustrating the detected ncBAF complex subunits upon co-immunoprecipitation and subsequent mass spectrometry analysis with the regular or alternative BRD9 isoforms stably overexpressed in the HEK293T cell line. The relative protein levels are based on the total number of identified peptide-spectrum matches (PSMs) for the corresponding protein from the mass spectrometry analysis. The ncBAF complex model to the right of the heatmap illustrates that the alternative BRD9 isoform precipitated a majority of the ncBAF complex subunits. ACTB was not detected among the co-immunoprecipitates. The control cells expressing FLAG-V5-tagged YFP were used. D Volcano plot displaying differential interaction analysis of proteins that selectively interacted more with the regular or alternative BRD9 isoforms (multiple Student’s t -tests with Benjamini–Hochberg multiple testing correction). The top 5 candidates are highlighted in red. E Venn diagram showing a comparison of the significant differentially interacting proteins with the regular or alternative BRD9 isoforms and BRD9-interacting proteins reported by Gaudio et al. . The intersecting proteins are SPEN, BRCA2, and CHD9. F Western blot analysis of V5-tag and BICRA co-immunoprecipitates in the HEK293T cell line with stable overexpression of the regular and alternative BRD9 isoforms. In the V5-tag-immunoprecipitations, both overexpressed BRD9 isoforms precipitated BICRA together with SMARCA4 and SMARCC1. Reciprocal BICRA co-immunoprecipitations precipitated both overexpressed BRD9 isoforms, SMARCA4 and SMARCC1. The control cells were transduced with an empty lentiviral vector. For complete blot images see Supplementary Fig. . G Bar plots showing V5-tag and BICRA co-immunoprecipitation efficiency with BRD9, BICRA, SMARCA4, and SMARCC1. Results are expressed as fold differences between the regular and alternative BRD9 isoforms with corresponding P values (Student’s t -test). The bar plots display the mean values from three repeated experiments, with error bars representing the standard deviation. For detailed quantification and calculation see Supplementary Fig. . PSM: peptide-spectrum matches; FDR: false discovery rate; IP: immunoprecipitation.

Article Snippet: Cell lines and primary CLL cells were treated with BRD9 inhibitors/degraders, I-BRD9 (SML1534; Sigma-Aldrich), PROTAC BRD9 Degrader-1 (HY-103632; MedChemExpress, New Jersey, NJ, USA), and dBRD9 Hydrochloride (SML2911; Sigma-Aldrich) [ , ].

Techniques: Sequencing, Construct, Over Expression, Immunoprecipitation, Mass Spectrometry, Stable Transfection, Control, Expressing, Comparison, Western Blot, Transduction, Plasmid Preparation, Standard Deviation

Journal: Leukemia

Article Title: The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1 -mutated chronic lymphocytic leukemia

doi: 10.1038/s41375-024-02379-4

Figure Lengend Snippet: A Volcano plot depicting differentially expressed genes between 18 SF3B1 MUT and 17 SF3B1 WT subset #2 cases with 22 downregulated and 55 upregulated genes ( | log 2 FC | ≥ 0.58 and FDR < 0.01). B Unsupervised clustermap illustrating the distinct clustering of SF3B1 MUT and SF3B1 WT subset #2 cases based on differential gene expression. The four neighboring genes, NOL9 , TAS1R1 , ZBTB48 , and KLHL21 , located on chromosome 1 are depicted in color. The Ward method and the Euclidean metric were employed. C Karyoplot of chromosome 1 displaying differentially expressed genes between SF3B1 MUT and SF3B1 WT subset #2 cases. Examination of chromosomal positions revealed four neighboring genes, NOL9 , TAS1R1 , ZBTB48 , and KLHL21 . D Venn diagram showing the overlap of genes with differential expression between SF3B1 MUT and SF3B1 WT cases in three independent datasets; subset #2 CLL, ICGC CLLE-ES [ , ], and TCGA CLL [ , , ]. For the differential gene expression analyses of the latter two datasets see Supplementary Fig. . ZBTB48 appeared upregulated in all three datasets, while TAS1R1 in subset #2 CLL and ICGC CLLE-ES. E Venn diagram showing the overlap of genes with higher expression levels in SF3B1 MUT subset #2 cases and increased chromatin accessibility in SF3B1 MUT CLL cases from Beekman et al. . The overlap comprises NOL9 , TAS1R1 , ZBTB48 , and KLHL21 . F Coverage tracks from ChIP-seq data sourced from the ENCODE database [ , ], based on the CML cell line K562, illustrating the binding of BRD9 and the ncBAF complex subunits SMARCA4 and SMARCC2 to the gene quartet region on chromosome 1. The promoters for these genes appear active based on the signal for H3K27ac, H3K4me3, H3K9ac, and H3K79me2, and transcriptionally active based on the H3K36me3 signal, whereas transcriptional repression is low based on the low signal for H3K27me3 and H3K9me3. G Gene essentiality map showing the relationship between efficacy and selectivity of genes in shinyDepMap [ , ]. SF3B1 appears as a non-selective dependency with high efficacy, while BRD9 as a selective dependency with high efficacy. BRD9 is in the dependency region of PIK3R1 , EZH2 , and FBXW7 . H Functional similarity clustering showing a chromatin remodeling dependency cluster connected to the BRD9 dependency signature in shinyDepMap. BRD9 clusters with SMARCD1 , a BAF complex subunit, subunits of the Mediator complex, p300/CBP subunits, and subunits of the SAGA complex. I BRD9 dependency ranking of the 1086 DepMap (22Q2) cell lines. For CLL cell lines, gene effect scores are given within the parenthesis. WT: wildtype; MUT: mutated; FDR: false discovery rate; FC: fold change.

Article Snippet: Cell lines and primary CLL cells were treated with BRD9 inhibitors/degraders, I-BRD9 (SML1534; Sigma-Aldrich), PROTAC BRD9 Degrader-1 (HY-103632; MedChemExpress, New Jersey, NJ, USA), and dBRD9 Hydrochloride (SML2911; Sigma-Aldrich) [ , ].

Techniques: Gene Expression, Quantitative Proteomics, Expressing, ChIP-sequencing, Binding Assay, Functional Assay

Journal: Leukemia

Article Title: The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1 -mutated chronic lymphocytic leukemia

doi: 10.1038/s41375-024-02379-4

Figure Lengend Snippet: A Dose-response analysis of I-BRD9, PROTAC BRD9 Degrader-1, and dBRD9 treatments in 3 SF3B1 WT cell lines, MEC1, PGA1, and HG3 (all CLL), and 3 SF3B1 MUT cell lines, CII (CLL), HNT34 (AML), and MEL202 (UVM). The cell lines were treated with drug concentrations ranging from 0.001 to 50 µM for 3 days, and cell viability was determined by CellTiter-Glo 2.0. Complete cell killing was exclusively observed with I-BRD9 treatment, allowing for the determination of corresponding IC 50 values for each cell line. The HNT34 cell line exhibited sensitivity to all three drugs, thereby allowing for the determination of IC 50 values for all conditions. Dose-response curves are shown with 95% confidence intervals, while individual dots display the mean values of triplicates, with error bars representing the standard deviation. B I-BRD9 sensitivity profile in 958 cancer cell lines from GDSC . C Assessment of proliferation in an SF3B1 WT cell line, PGA1, and SF3B1 MUT cell lines, CII and HNT34, upon treatment with 10 µM I-BRD9, 25 µM PROTAC BRD9 Degrader-1, or 25 µM dBRD9. Cell lines were treated for 3 days with a 5-hour exposure to BrdU at the end, and proliferation was quantified by flow cytometry, measured as BrdU+ cells. Vehicle (DMSO) was used as the negative control for drug treatment, while cells not exposed to BrdU served as the negative control to assess the specificity of the anti-BrdU antibody. The upper left quadrants (BrdU+ cells) in the density plots represent the percentages of proliferating cells. D Bar plot displaying differences in the percentages of proliferating cells compared to negative controls with corresponding P values (one-way ANOVA). The bar plot displays the mean values from two repeated experiments, with error bars representing the standard deviation. E Assessment of apoptosis in the same cell lines and the identical samples as in ( C ). Treatment with 5 µM Camptothecin and vehicle (DMSO) were used as the positive and negative controls, respectively. Apoptosis was evaluated by Annexin V/PI staining and subsequent flow cytometry analysis. The lower left (Annexin V-/PI- cells), upper left (Annexin V+/PI- cells), and upper right (Annexin V+/PI+ cells) quadrants in the density plots represent the percentages of viable, early apoptotic, and late apoptotic cells, respectively. F Stacked bar plot displaying differences in the percentages of viable, early apoptotic, and late apoptotic cells compared to negative controls with corresponding P values (one-way ANOVA). The bar plot displays the mean values from two repeated experiments, with error bars representing the standard deviation. G Bar plot showing cell viability differences as determined by CellTiter-Glo 2.0 in the same cell lines under the same experimental conditions as in panels ( C , E ) with corresponding P values (one-way ANOVA). The bar plot displays the mean values from two repeated experiments in triplicates, with error bars representing the standard deviation. WT: wildtype; MUT: mutated; IC 50 : half-maximal inhibitory concentration; RLU: relative luminescence unit.

Article Snippet: Cell lines and primary CLL cells were treated with BRD9 inhibitors/degraders, I-BRD9 (SML1534; Sigma-Aldrich), PROTAC BRD9 Degrader-1 (HY-103632; MedChemExpress, New Jersey, NJ, USA), and dBRD9 Hydrochloride (SML2911; Sigma-Aldrich) [ , ].

Techniques: Standard Deviation, Flow Cytometry, Negative Control, Staining, Concentration Assay

Journal: Leukemia

Article Title: The non-canonical BAF chromatin remodeling complex is a novel target of spliceosome dysregulation in SF3B1 -mutated chronic lymphocytic leukemia

doi: 10.1038/s41375-024-02379-4

Figure Lengend Snippet: A Dose-response analysis of I-BRD9 treatment in 3 SF3B1 WT (SKL48, SKL152, SKL53) and 3 SF3B1 MUT (SKL147, SKL47, SKL157) primary CLL cell samples. The cell lines were treated with drug concentrations ranging from 0.001 to 50 µM for 2 days, and cell viability was determined by CellTiter-Glo 2.0. Dose-response curves are shown with 95% confidence intervals, while individual dots display the mean values of triplicates, with error bars representing the standard deviation. B Stacked density plots showing apoptosis assessment in the same primary CLL cell samples as in ( A ). Treatment with 5 µM Camptothecin and vehicle (DMSO) were used as the positive and negative controls, respectively. Apoptosis was evaluated by Annexin V/PI staining and subsequent flow cytometry analysis. The lower left (Annexin V-/PI- cells), upper left (Annexin V+/PI- cells), and upper right (Annexin V+/PI+ cells) quadrants in the density plots represent the percentages of viable, early apoptotic, and late apoptotic cells, respectively. C Stacked bar plot displaying differences in the percentages of viable, early apoptotic, and late apoptotic cells compared to negative controls with corresponding P values (Student’s t -test). The bar plot displays the mean values, with error bars representing the standard deviation. WT: wildtype; MUT: mutated; IC 50 : half-maximal inhibitory concentration; RLU: relative luminescence unit.

Article Snippet: Cell lines and primary CLL cells were treated with BRD9 inhibitors/degraders, I-BRD9 (SML1534; Sigma-Aldrich), PROTAC BRD9 Degrader-1 (HY-103632; MedChemExpress, New Jersey, NJ, USA), and dBRD9 Hydrochloride (SML2911; Sigma-Aldrich) [ , ].

Techniques: Standard Deviation, Staining, Flow Cytometry, Concentration Assay

Journal: Nature Communications

Article Title: The bromodomain containing protein BRD-9 orchestrates RAD51–RAD54 complex formation and regulates homologous recombination-mediated repair

doi: 10.1038/s41467-020-16443-x

Figure Lengend Snippet: a Signature analysis of six BRD-containing proteins that have been associated with homologous recombination (HR)-related mutation signatures (signature 3) in ovarian cancer in the TCGA database. p Values were calculated by one-sided Fisher Exact test. b Quantification of HR- and NHEJ-mediated DSB repair as assessed using GFP reporter assay in HCT-116 cells following knockdown of bromodomain-containing proteins. The indicated bromodomain-containing proteins were individually knocked down in HCT-116 cells transfected with GFP-tagged reporter plasmid. Thirty-six hours later, repair efficiency was assessed using flow cytometry. The BRCA1- and 53BP1-knockdown cells were used as positive control for HR and NHEJ, respectively. Representative data (mean ± SEM) are shown from n = 3 biologically independent samples; * p < 0.05, ** p < 0.005, *** p < 0.001 by two-sided unpaired t test. c , d Knockdown of BRD9 causes HR but not NHEJ deficiency. OVCAR8 cells were infected with lentivirus expressing the indicated BRD9 shRNAs. Thirty-six hours later, HR- ( c ) and NHEJ- ( d ) mediated repair capacity was assessed using flow cytometry. The BRCA1 and 53BP1 shRNAs were used as positive controls for HR and NHEJ, respectively. Representative data (mean ± SEM) are shown from n = 3 biologically independent samples. * p < 0.05, ** p < 0.01, *** p < 0.001 by two-sided unpaired t test. e , f BRD9 inhibitor (I-BRD9) selectively inhibits HR and not NHEJ activity. OVCAR8 cells were treated with 10 or 20 µM I-BRD9 for 36 h, and then subjected to HR ( e ) and NHEJ ( f ) assay as described in c , d . Representative data (mean ± SEM) are shown from n = 3 biologically independent samples. * p < 0.05, ** p < 0.01, *** p < 0.001 by two-sided unpaired t test. g – j Knockdown of BRD9 delays clearance of γ-H2AX and RAD51 foci. OVCAR8 cells were infected with lentivirus-expressing control (Ctrl) or BRD9 shRNA. Cells were exposed to 2-Gy irradiation and fixed at the indicated time points. Cells were stained for the indicated foci. g , i Representative images are shown of γ-H2AX and Rad51 foci after the indicated treatments and indicated time following 2-Gy irradiation. h , j γ-H2AX and Rad51 foci in OVCAR8 cells after the indicated treatment and time following 2-Gy irradiation were quantified. Representative data (mean ± SEM) are shown from three independent experiments. *** p < 0.001 by two-sided unpaired t test. Scale bar, 10 μm.

Article Snippet: The RAD51 and GFP BRD9 (65380) were purchased from Addgene and subcloned into plvx3 or CMV-HA vector (635690 Clontech). pEGFP-Rad54-N1 plasmid was provided by Christel Braun lab. Rad54-GST plasmid was provided by Wolf-Dietrich Heyer lab.

Techniques: Homologous Recombination, Mutagenesis, Reporter Assay, Knockdown, Transfection, Plasmid Preparation, Flow Cytometry, Positive Control, Infection, Expressing, Activity Assay, Control, shRNA, Irradiation, Staining

Journal: Nature Communications

Article Title: The bromodomain containing protein BRD-9 orchestrates RAD51–RAD54 complex formation and regulates homologous recombination-mediated repair

doi: 10.1038/s41467-020-16443-x

Figure Lengend Snippet: a – d Representative immunofluorescence images of OVCAR8 cells demonstrating that BRD9 knockdown reduces RAD54 foci formation and RAD51/RAD54 co-localization. a – c OVCAR8 cells were infected with lentivirus-expressing control (Ctrl) or BRD9 shRNA and exposed to 2-Gy IR. Eight hours later, cells were stained with RAD51 (red) and RAD54 (green) antibodies. Representative immunofluorescence images are shown in a . RAD51 foci/cell ( b ), RAD54 foci/cell ( c ), and RAD51/RAD54 co-localized foci ( d ) after the indicated treatment were quantified. Representative data (mean ± SEM) are shown from three independent experiments. n = 50 cells examined over three independent experiments. *** p < 0.001 by two-sided unpaired t test. Scale bar, 10 μm. e , f DNA damage-induced RAD51–RAD54 interaction is dependent on BRD9. Endogenous BRD9 was knocked out in HEK293T cells using shRNA, and the indicated constructs were expressed. Cells were exposed to 10-Gy irradiation. Lysates were collected after 8 h, and immunoprecipitation with the indicated antibodies was performed. Blots were probed with the indicated antibodies. g RAD51 is necessary for BRD9 foci formation. OVCAR8 cells were infected with lentivirus-expressing RAD51 shRNA, exposed to 2-Gy IR, and fixed after 8 h. Cells were stained with BRD9 (green) and RAD51 (red) antibodies. Representative images of the indicated foci are shown. BRD9 foci/cell were quantified. Representative data (mean ± SEM) are shown from three independent experiments. n = 50 cells examined over three independent experiments. *** p < 0.001 by two-sided unpaired t test. Scale bar, 10 μm.

Article Snippet: The RAD51 and GFP BRD9 (65380) were purchased from Addgene and subcloned into plvx3 or CMV-HA vector (635690 Clontech). pEGFP-Rad54-N1 plasmid was provided by Christel Braun lab. Rad54-GST plasmid was provided by Wolf-Dietrich Heyer lab.

Techniques: Immunofluorescence, Knockdown, Infection, Expressing, Control, shRNA, Staining, Construct, Irradiation, Immunoprecipitation

Journal: Nature Communications

Article Title: The bromodomain containing protein BRD-9 orchestrates RAD51–RAD54 complex formation and regulates homologous recombination-mediated repair

doi: 10.1038/s41467-020-16443-x

Figure Lengend Snippet: a , b BRD9 interacts with RAD54 and RAD51. a 293T cells were transfected with HA-tagged BRD9 plasmid. Forty-eight hours after transfection, cells were exposed to 10-Gy IR and collected at the indicated time points. Immunoprecipitation with anti-HA beads was performed. Blots were probed with the indicated antibodies. b OVCAR8 cells were exposed to 10-Gy IR. Lysates were collected after 8 h, and co-immunoprecipitation with anti-BRD9 antibody was performed. Blots were probed with the indicated antibodies. c – g BRD9 interacts with RAD51 through the C terminus, and its bromodomain mediates its interaction with RAD54. c Schematic diagram depicting a set of HA-tagged BRD9 expression constructs. d 293T cells were transfected with the indicated constructs of HA-BRD9 and exposed to 10-Gy IR. Lysates were collected after 8 h. Immunoprecipitation with anti-HA beads was performed. Blots were probed with the indicated antibodies. e 293T cells were transfected with the indicated constructs. Forty-eight hours later, cells were treated with 10-Gy IR 8 h before immunoprecipitation with anti-HA beads. Blots were probed with indicated antibodies. f , g 293T cells were transfected with the indicated constructs. Twenty-four hours later, cells were exposed to BRD9i, 10-Gy IR, or both. Lysates were collected after 8 h. Immunoprecipitation with anti-HA beads was performed. Blots were probed with the indicated antibodies. h , k Bromodomain of BRD9 is essential for its HR activity. h BRD9-knockdown 293T cells were infected with the indicated lentiviral plasmids. Twenty-four hours after transfection, cells were exposed to IR. Lysates were collected after 8 h, and immunoprecipitation with anti-HA beads was performed. Blots were probed with the indicated antibodies. i BRD9-knockdown 293T cells expressing the DR-GFP reporter were infected with lentivirus expressing the indicated proteins. Twenty-four hours later, cells were subjected to flow cytometry. Representative data (mean ± SEM) are shown from n = 3 biologically independent samples. * p < 0.05, *** p < 0.001 by two-sided unpaired t test. j , k BRD9-knockdown OVCAR8 cells were infected with lentivirus expressing the indicated proteins, and subjected to colony-formation assay to assess the sensitivity to PARPi ( j ) and cisplatin ( k ). Representative data (mean ± SEM) are shown from n = 3 biologically independent samples. * p < 0.05, ** p < 0.01, *** p < 0.001 by two-sided unpaired t test.

Article Snippet: The RAD51 and GFP BRD9 (65380) were purchased from Addgene and subcloned into plvx3 or CMV-HA vector (635690 Clontech). pEGFP-Rad54-N1 plasmid was provided by Christel Braun lab. Rad54-GST plasmid was provided by Wolf-Dietrich Heyer lab.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Construct, Activity Assay, Knockdown, Infection, Flow Cytometry, Colony Assay

Journal: Nature Communications

Article Title: The bromodomain containing protein BRD-9 orchestrates RAD51–RAD54 complex formation and regulates homologous recombination-mediated repair

doi: 10.1038/s41467-020-16443-x

Figure Lengend Snippet: a – c BRD9 is overexpressed in ovarian cancer patient samples and cell lines, and is associated with decreased patient survival. a Analysis of BRD9 expression in ovarian cancer patient samples from the TCGA database. Red indicates BRD9 amplification. b Kaplan–Meier curve showing the survival of ovarian cancer patients with low ( n = 720) and high ( n = 715) expression of BRD9. Statistical analysis with the two-sided log-rank (Mantel–Cox) test revealed statistically significant differences as shown on the graph. c Immunoblot of indicated proteins in the indicated ovarian cancer cell lines. d – g Depletion/inhibition of BRD9 sensitizes OVCAR8 cells to PARPi and cisplatin. d – f OVCAR8 cells infected with indicated shRNAs and subjected to WB assay ( d ) to assess knockdown efficiency, and colony-formation assay to assess the sensitivity to olaparib ( e ) and cisplatin ( f ). g OVCAR8 cells were exposed to olaparib and/or BRD9i and subjected to colony-formation assay. Representative data (mean ± SEM) are shown from n = 3 biologically independent samples. ** p < 0.01,*** p < 0.001 by two-sided unpaired t test. h – j Overexpression of BRD9 confers resistance to PARPi/cisplatin. OVCAR7 cells infected with the indicated lentivirus and subjected to colony-formation assay to assess the sensitivity to olaparib ( i ) and cisplatin ( j ). Protein expression was assessed by Western blot ( h ). Representative data (mean ± SEM) are shown from n = 3 biologically independent samples. ** p < 0.01,*** p < 0.001 by two-sided unpaired t test. k , l Depletion/inhibition of BRD9 sensitizes OVCAR8 cells to PARPi in vivo. k OVCAR8 cells expressing the indicated shRNAs were subcutaneously injected into the flank of NOD-SCID mice. Mice were treated with or without olaparib (50 mg/kg i.p. 3 days × 8 times). Tumor volume and weight were assessed ( l ). OVCAR8 cells were subcutaneously injected into the flank of NOD-SCID mice. Mice were treated with vehicle (normal saline, NS), olaparib (PARPi, 50 mg/kg), BRD9i (40 mg/kg i.p. 3 days × 8 times), or olaparib + BRD9i. Tumor volume and weight were assessed. Representative data (mean ± SEM) are shown from n = 6 biologically independent samples by two-sided unpaired t test.

Article Snippet: The RAD51 and GFP BRD9 (65380) were purchased from Addgene and subcloned into plvx3 or CMV-HA vector (635690 Clontech). pEGFP-Rad54-N1 plasmid was provided by Christel Braun lab. Rad54-GST plasmid was provided by Wolf-Dietrich Heyer lab.

Techniques: Expressing, Amplification, Western Blot, Inhibition, Infection, Knockdown, Colony Assay, Over Expression, In Vivo, Injection, Saline

Journal: Nature Communications

Article Title: The bromodomain containing protein BRD-9 orchestrates RAD51–RAD54 complex formation and regulates homologous recombination-mediated repair

doi: 10.1038/s41467-020-16443-x

Figure Lengend Snippet: Bromodomain-containing protein–HR signature.

Article Snippet: The RAD51 and GFP BRD9 (65380) were purchased from Addgene and subcloned into plvx3 or CMV-HA vector (635690 Clontech). pEGFP-Rad54-N1 plasmid was provided by Christel Braun lab. Rad54-GST plasmid was provided by Wolf-Dietrich Heyer lab.

Techniques: Mutagenesis

Journal: Cancers

Article Title: Synergy between BRD9- and IKZF3-Targeting as a Therapeutic Strategy for Multiple Myeloma

doi: 10.3390/cancers16071319

Figure Lengend Snippet: BRD9 inhibitor or degrader treatment potentiates effects of IMiDs on MM cell proliferation. ( A , B ) Proliferation assays: Effects of lenalidomide ( A ) or pomalidomide ( B ) alone or combined with the BRD9 inhibitor, EA-89, against H929 cells following 4 days. ( C ) Proliferation assay: Effects of pomalidomide alone or combined with the BRD9 degrader, QA-68, against H929 cells following 4 days. ( D ) Proliferation assay: Effects of pomalidomide alone or combined with EA-89 against 8226 cells following 6 days. Results shown here are representative of additional studies for which similar results were observed. ( E ) Combination indices generated by Calcusyn software for proliferation studies investigating combinations of BRD9-targeting agents and IMiDs. ( F , G ) Treatment of normal PBMCs following 4 days ( F ) or CD45+ leukocyte sub-population of PBMCs following 5 days ( G )treatment with pomalidomide alone, EA-89 alone, or a combination of both agents ( n = 6). Paired t -tests showing statistical significance p < 0.05 are denoted by * between the biological treatment groups EA89 alone and pomalidomide alone/combination but no significance observed between pomalidomide alone and combination.

Article Snippet: GAPDH antibody (14C10) (rabbit mAb, #2118), c-MYC antibody (D84C12) (rabbit mAb, #5605), Ikaros antibody (D6N9Y) (rabbit mAb, #14859), Aiolos antibody (D1C1E) (rabbit mAb, #15103), BRD9 antibody (E9R21) (rabbit mAb, #58906), and CRBN antibody (D8H3S) (rabbit mAb, #71810) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Proliferation Assay, Generated, Software

Journal: Cancers

Article Title: Synergy between BRD9- and IKZF3-Targeting as a Therapeutic Strategy for Multiple Myeloma

doi: 10.3390/cancers16071319

Figure Lengend Snippet: Effects of treatment of MM cells with IMiDs or BRD9 inhibitor or degrader, alone or in combination, on Ikaros, MYC, and CRBN protein levels. ( A ) Immunoblots: Effects of pomalidomide or EA-89 alone or combined on IKZF1, IKZF3, MYC, and CRBN protein levels following 24 h. ( B – E ) Immunoblots: Effect of lenalidomide ( B ), EA-89 ( C ), QA-68 ( D ), and pomalidomide ( E ) on levels of IKZF3 and MYC protein levels following 24 h. CRBN protein levels were analyzed in EA-89-treated cells ( C ). The uncropped blots are shown in .

Article Snippet: GAPDH antibody (14C10) (rabbit mAb, #2118), c-MYC antibody (D84C12) (rabbit mAb, #5605), Ikaros antibody (D6N9Y) (rabbit mAb, #14859), Aiolos antibody (D1C1E) (rabbit mAb, #15103), BRD9 antibody (E9R21) (rabbit mAb, #58906), and CRBN antibody (D8H3S) (rabbit mAb, #71810) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot

Journal: Cancers

Article Title: Synergy between BRD9- and IKZF3-Targeting as a Therapeutic Strategy for Multiple Myeloma

doi: 10.3390/cancers16071319

Figure Lengend Snippet: BRD9 KD potentiates effects of pomalidomide against growth of MM cells. ( A ) Proliferation assay: Effects of doxycycline-induced BRD9 KD on growth of H929 cells. For ( A ), asterisks indicate statistical significance ( p value < 0.05). ( B ) Immunoblot: Measurement of BRD9 KD efficiency in H929 BRD9 KD#1, BRD9 KD#2, and BRD9 KD#5 cell lines versus H929 control cells. Doxycycline-inducible BRD9 KD H929 cells (KD#1, KD#2, and KD#5) were compared with H929 control cells in terms of growth response to treatment with doxycycline plus or minus pomalidomide across the indicated range of concentrations for 2 days, 3 days, and 4 days. For ( C , D ), asterisks indicate statistical significance ( p value < 0.05). Results shown ( C , D ) are representative of other studies for which similar results were observed. The uncropped blots are shown in .

Article Snippet: GAPDH antibody (14C10) (rabbit mAb, #2118), c-MYC antibody (D84C12) (rabbit mAb, #5605), Ikaros antibody (D6N9Y) (rabbit mAb, #14859), Aiolos antibody (D1C1E) (rabbit mAb, #15103), BRD9 antibody (E9R21) (rabbit mAb, #58906), and CRBN antibody (D8H3S) (rabbit mAb, #71810) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Proliferation Assay, Western Blot, Control

Journal: Cancers

Article Title: Synergy between BRD9- and IKZF3-Targeting as a Therapeutic Strategy for Multiple Myeloma

doi: 10.3390/cancers16071319

Figure Lengend Snippet: GSEA enrichment analysis and PCR validation of MYC and E2F target genes. ( A ) GSEA enrichment analysis of RNA-seq results for H929 BRD9 KD#5 cells treated with doxycycline, pomalidomide, or a combination following 24 h. ( B ) Heatmap showing representative MYC and E2F targets downregulated in RNA-seq from the three sets (namely, doxycycline, pomalidomide, or a combination) in H929 BRD9 KD#1, 2, and 5 cells. ( C ) PCR validation of selected MYC and E2F target genes associated with BRD9 KD plus pomalidomide treatment in H929 BRD9 KD#2 cells following 24 h.

Article Snippet: GAPDH antibody (14C10) (rabbit mAb, #2118), c-MYC antibody (D84C12) (rabbit mAb, #5605), Ikaros antibody (D6N9Y) (rabbit mAb, #14859), Aiolos antibody (D1C1E) (rabbit mAb, #15103), BRD9 antibody (E9R21) (rabbit mAb, #58906), and CRBN antibody (D8H3S) (rabbit mAb, #71810) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Biomarker Discovery, RNA Sequencing

Journal: Cancers

Article Title: Synergy between BRD9- and IKZF3-Targeting as a Therapeutic Strategy for Multiple Myeloma

doi: 10.3390/cancers16071319

Figure Lengend Snippet: Inhibitory effects of pomalidomide combined with targeted loss of BRD9 on signaling pathways in MM. ( A , B , D , E ) Pathway analyses showing signaling pathways downregulated by doxycycline-induced BRD9 KD+pomalidomide treatment of H929 cells (clones #2 and #5) for 24 h. The full list of genes downregulated in response to doxycycline+pomalidomide was used for these analyses. Gene numbers are shown in parentheses. For ( A , B ), GeneCodis was utilized for pathway analysis, using GO Biological Process, and for ( D , E ), GeneCodis was utilized for pathway analysis, using Transcription Factors. ( C ) Validation (via qPCR) performed for EGR1, INHBE, and EGR2, downregulated in response to pomalidomide+doxycycline-induced BRD9 KD in H929 cells. Graphs shown are representative of replicates for which similar results were observed (KD#1 (EGR1, n = 2; INHBE, n = 1); KD#2 (EGR1, n = 2; EGR2, n = 2); KD#5 (EGR1, n = 2; INHBE, n = 1). Treatments were as follows: DMSO vehicle, doxycycline (1 μg/mL), pomalidomide (50 nM), and combination of doxycycline (1 μg/mL)+pomalidomide (50 nM). For doxycycline-treated H929 doxycycline-inducible BRD9 KD#1 cells, BRD9 KD#2 cells, and BRD9 KD#5 cells, BRD9 was downregulated Log2-fold −1.14, Log2-fold −0.53, and Log2-fold −1.02, respectively. MYC target genes include E2F transcription factors.

Article Snippet: GAPDH antibody (14C10) (rabbit mAb, #2118), c-MYC antibody (D84C12) (rabbit mAb, #5605), Ikaros antibody (D6N9Y) (rabbit mAb, #14859), Aiolos antibody (D1C1E) (rabbit mAb, #15103), BRD9 antibody (E9R21) (rabbit mAb, #58906), and CRBN antibody (D8H3S) (rabbit mAb, #71810) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Protein-Protein interactions, Clone Assay, Biomarker Discovery

Journal: Cancers

Article Title: Synergy between BRD9- and IKZF3-Targeting as a Therapeutic Strategy for Multiple Myeloma

doi: 10.3390/cancers16071319

Figure Lengend Snippet: Stimulatory effects of pomalidomide combined with targeted loss of BRD9 on signaling pathways in MM. ( A , B , D , E ) Pathway analyses showing signaling pathways upregulated by doxycycline-induced BRD9 KD+pomalidomide treatment of H929 cells (clones #1 and #5) for 24 h. The full list of genes downregulated in response to doxycycline+pomalidomide was used for these analyses. Gene numbers are shown in parentheses. For ( A , B ), GeneCodis was utilized for pathway analysis, using GO Biological Process, and for ( D , E ), GeneCodis was utilized for pathway analysis, using Transcription Factors. ( C ) Validation (via qPCR) performed for GIMAP4, upregulated in response to pomalidomide+doxycycline-induced BRD9 KD in H929 cells. Treatments were as follows: DMSO vehicle (24 h), doxycycline (1 μg/mL), pomalidomide (50 nM), and combination of doxycycline (1 μg/mL)+pomalidomide (50 nM). For doxycycline-treated H929 doxycycline-inducible BRD9 KD#1 cells, BRD9 KD#2 cells, and BRD9 KD#5 cells, BRD9 was downregulated Log2-fold −1.14, Log2-fold −0.53, and Log2-fold −1.02, respectively.

Article Snippet: GAPDH antibody (14C10) (rabbit mAb, #2118), c-MYC antibody (D84C12) (rabbit mAb, #5605), Ikaros antibody (D6N9Y) (rabbit mAb, #14859), Aiolos antibody (D1C1E) (rabbit mAb, #15103), BRD9 antibody (E9R21) (rabbit mAb, #58906), and CRBN antibody (D8H3S) (rabbit mAb, #71810) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Protein-Protein interactions, Clone Assay, Biomarker Discovery

Journal: Cancers

Article Title: Synergy between BRD9- and IKZF3-Targeting as a Therapeutic Strategy for Multiple Myeloma

doi: 10.3390/cancers16071319

Figure Lengend Snippet: Overriding IMiD resistance with BRD9 targeting combined with IMiD treatment. ( A ) Proliferation assay: Effects of iberdomide against parental H929 cells (treated with DMSO for 4 months) or IMiD-resistant H929 cells (treated for 4 months with 10–20 nM iberdomide) following 5 days. ( B ) Immunoblots: (upper panel) Comparison of levels of CRBN, MYC, and IKZF3 protein in parental H929 cells (treated with DMSO for 4 months) or IMiD-resistant#2 H929 cells (treated for 4 months with 10 nM iberdomide); (lower panel) Effects of EA-89 (3000 nM) treatment of H929 iber-domide-resistant cells on CRBN levels following 24 h. ( C ) Proliferation assay: Effects of pomalidomide against parental H929 cells (treated with DMSO for 4 months) or IMiD-resistant H929 cells (treated for 4 months with 10–20 nM iberdomide) following 4 days. ( D ) Proliferation assays: (left panel) Effects of pomalidomide alone or combined with the EA-89 against H929 cells (treated with DMSO for 4 months) following 4 days. (right panel) Effects of pomalido-mide alone or combined with the EA-89 against H929 IMiD-resistant H929 cells (treated for 4 months with 10–20 nM iberdomide) following 4 days. The uncropped blots are shown in .

Article Snippet: GAPDH antibody (14C10) (rabbit mAb, #2118), c-MYC antibody (D84C12) (rabbit mAb, #5605), Ikaros antibody (D6N9Y) (rabbit mAb, #14859), Aiolos antibody (D1C1E) (rabbit mAb, #15103), BRD9 antibody (E9R21) (rabbit mAb, #58906), and CRBN antibody (D8H3S) (rabbit mAb, #71810) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Proliferation Assay, Western Blot, Comparison

Journal: bioRxiv

Article Title: The PBAF chromatin remodeling complex contributes to metal homeostasis through Mtf1 regulation

doi: 10.1101/2025.04.12.648552

Figure Lengend Snippet: (A-C) Representative western blots (top) and quantification (bottom) of Baf180 (A) , Baf250a (B) , and Brd9 (C) protein levels in proliferating myoblasts, normalized to GAPDH as a loading control. Data represents the mean ± SE of three independent biological replicates. *P < 0.05 compared to scr control. (D-F) Proliferation analysis of WT, scr, and KD myoblasts over 72 h using cell counting assays for cells cultured under basal conditions (D) or supplemented with 100 µM CuSO 4 (E) or 50 µM ZnSO 4 (F) . Baf180 KD myoblasts exhibits no significant difference under basal conditions, non-treated (NT), but this cell line sensitive to both metals. Baf250 KD and Brd9 KD show reduced proliferation, which is rescued by metal supplementation. Data represent the mean ± SE of three independent experiments. *P < 0.05; **P < 0.01 compared to the same strain cultured in the absence of metals (NT).

Article Snippet: Rabbit anti-Baf180 (89123S), -Baf250a (12354S), and -Brd9 (48306S), used for confocal microscopy at dilutions of 1:50, 1:50, and 1:100 respectively, were from Cell Signaling Technology.

Techniques: Western Blot, Control, Cell Counting, Cell Culture

Journal: bioRxiv

Article Title: The PBAF chromatin remodeling complex contributes to metal homeostasis through Mtf1 regulation

doi: 10.1101/2025.04.12.648552

Figure Lengend Snippet: Flame atomic absorption spectroscopy (AAS) analysis quantifying total intracellular Cu and Zn levels in C2C12 myoblasts under different conditions. (A) Quantification of total Cu and (B) total Zn for proliferating myoblasts cultured in basal medium without supplementation. (C) Quantification of total Cu and (D) total Zn for proliferating myoblasts cultured in the presence of 100 μM CuSO 4 . (E) Quantification of total Cu and (F) total Zn for proliferating myoblasts cultured with 50 μM ZnSO 4 . Baf180 KD myoblasts exhibit significantly higher intracellular Cu accumulation upon CuSO 4 supplementation, suggesting impaired Cu homeostasis. Baf250A KD have lower Cu and Zn content while Brd9 KD myoblasts showed reduced levels of Zn. These defects are restored upon metal supplementation. Data represents the mean ± SE of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 compared to scr control.

Article Snippet: Rabbit anti-Baf180 (89123S), -Baf250a (12354S), and -Brd9 (48306S), used for confocal microscopy at dilutions of 1:50, 1:50, and 1:100 respectively, were from Cell Signaling Technology.

Techniques: Atomic Absorption Spectroscopy, Cell Culture, Control

Journal: bioRxiv

Article Title: The PBAF chromatin remodeling complex contributes to metal homeostasis through Mtf1 regulation

doi: 10.1101/2025.04.12.648552

Figure Lengend Snippet: Confocal microscopy live-cell analysis of labile Cu levels in wild-type (A) , Scr control (B) , and KD myoblasts for Baf180 (C) , Baf250a (D) , and Brd9 (E) under cultured for 48h in basal untreated media (NT) or supplemented with 100 μM CuSO 4 or 50 μM ZnSO 4. Labile Cu⁺ (green) was detected using CS1, while Cu²⁺ (red) was visualized using CD649.2. F. Quantification of the fluorescence of live-cell imaging for Cu + with the CS1 probe (left panel) and Cu 2+ with the CD649.2 probe (right panel) from proliferating C2C12 myoblasts. N = 3, *P < 0.05; ** P < 0.01, ***P < 0.001; ****P < 0.0001. Baf180 KD myoblasts exhibited an decreased pools of labile Cu + under all conditions tested, suggesting impaired copper mobilization. Scale bar: 10 μm.

Article Snippet: Rabbit anti-Baf180 (89123S), -Baf250a (12354S), and -Brd9 (48306S), used for confocal microscopy at dilutions of 1:50, 1:50, and 1:100 respectively, were from Cell Signaling Technology.

Techniques: Confocal Microscopy, Cell Analysis, Control, Cell Culture, Fluorescence, Live Cell Imaging

Journal: bioRxiv

Article Title: The PBAF chromatin remodeling complex contributes to metal homeostasis through Mtf1 regulation

doi: 10.1101/2025.04.12.648552

Figure Lengend Snippet: Representative confocal microscopy images showing the expression and nuclear localization of Baf180 (red) and Mtf1 (green) in wild type (A) , scr (B) , Baf180 KD (C) , Baf250a KD (D) and Brd9 KD (E) C2C12 myoblasts. Nuclei are counterstained with DAPI (blue). (F) Quantification of Mtf1 colocalization with Baf180 in control and the different SWI/SNF KD myoblasts from panel A-E and from supplemental figures 6 and 7. Colocalization is disrupted in Baf180 KD cells, while metal treatment modulates Mtf1-Baf180 interactions. Scale bars: 10 μm (panoramic views), 5 μm (zoomed-in images). Mean ± SE of three independent biological replicates. *P < 0.05, **P < 0.01, ****P < 0.0001. * Indicates significance relative to untreated WT cells. (G) Representative immunoprecipitation (IP) of Mtf1 or Baf180 in proliferating wild type myoblasts (N=3). Input, a Baf180 IP obtained from Baf180 KD cells and an IgG IP were included as controls. Mtf1 exhibits significantly higher colocalization with Baf180 compared to Baf250a or Brd9 in WT proliferating myoblasts. To show both proteins, the western blot was developed using both the rabbit anti-Baf180 and the mouse anti-Mtf1 specific antibodies, incubated sequentially in the same membrane, followed by sequential incubation of species-specific secondary antibodies.

Article Snippet: Rabbit anti-Baf180 (89123S), -Baf250a (12354S), and -Brd9 (48306S), used for confocal microscopy at dilutions of 1:50, 1:50, and 1:100 respectively, were from Cell Signaling Technology.

Techniques: Confocal Microscopy, Expressing, Control, Immunoprecipitation, Western Blot, Incubation, Membrane