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brd9 bromodomain inhibitor  (MedChemExpress)


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    MedChemExpress brd9 bromodomain inhibitor
    (A) Schematic showing how the Parbit expression cassette is used to generate stably expressed Acyl-eCRs in mESCs. RMCE by Cre, followed by a double selection of ganciclovir and puromycin, was applied to generate these constructs at a defined site in the mouse genome. A CAG promoter drives the constitutive expression of the <t>bromodomain</t> of interest, which is fused to a nuclear localization signal (NLS), and an eGFP tag. This construct also fuses a biotin acceptor site to the N-terminus of the protein, which can be biotinylated in vivo by a bacterial BirA ligase. (B) Schematic diagram showing how CBP was endogenously tagged with an eGFP tag. A homology donor construct was generated by cloning a 900 bp upstream and a 1,048 bp (CBP) or 1,068 bp (p300) downstream homology arm flanking a 30 bp flexible GGS linker that was fused to an eGFP tag. This donor construct was co-transfected with a pX330 CRISPR-Cas9 plasmid, which had an sgRNA targeting the C-terminus of the CBP gene. (C-D) Sanger sequencing of genotyping PCR products from the C-terminus of the CBP (C) and p300 (D) loci, confirming the in-frame homologous integration of an eGFP tag. Data shown are from mESC clone #1 for both CBP and p300 tagging. (E) Flow cytometry data showing the eGFP signal from cell lines where either CBP or p300 were endogenously tagged with eGFP. Two clonal replicates for each protein tagging are shown. Cell lines were maintained in culture for more than two weeks to demonstrate stable expression of the eGFP fusion proteins. (F) Western blot of nuclear extracts from cell lines treated with 1 μM dCBP-1 PROTAC for the stated duration. An antibody against GFP was used to probe the eGFP tag on the Acyl-eCR constructs. The CBP_BRD.1x eCR runs at approximately 60 kDa, and the Empty-eGFP construct runs at 33 kDa. Non-specific bands are marked by an asterisk (*). Revert700 Total Protein Stain is used to show equal loading in lanes.
    Brd9 Bromodomain Inhibitor, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains"

    Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

    Journal: bioRxiv

    doi: 10.1101/2025.09.06.674632

    (A) Schematic showing how the Parbit expression cassette is used to generate stably expressed Acyl-eCRs in mESCs. RMCE by Cre, followed by a double selection of ganciclovir and puromycin, was applied to generate these constructs at a defined site in the mouse genome. A CAG promoter drives the constitutive expression of the bromodomain of interest, which is fused to a nuclear localization signal (NLS), and an eGFP tag. This construct also fuses a biotin acceptor site to the N-terminus of the protein, which can be biotinylated in vivo by a bacterial BirA ligase. (B) Schematic diagram showing how CBP was endogenously tagged with an eGFP tag. A homology donor construct was generated by cloning a 900 bp upstream and a 1,048 bp (CBP) or 1,068 bp (p300) downstream homology arm flanking a 30 bp flexible GGS linker that was fused to an eGFP tag. This donor construct was co-transfected with a pX330 CRISPR-Cas9 plasmid, which had an sgRNA targeting the C-terminus of the CBP gene. (C-D) Sanger sequencing of genotyping PCR products from the C-terminus of the CBP (C) and p300 (D) loci, confirming the in-frame homologous integration of an eGFP tag. Data shown are from mESC clone #1 for both CBP and p300 tagging. (E) Flow cytometry data showing the eGFP signal from cell lines where either CBP or p300 were endogenously tagged with eGFP. Two clonal replicates for each protein tagging are shown. Cell lines were maintained in culture for more than two weeks to demonstrate stable expression of the eGFP fusion proteins. (F) Western blot of nuclear extracts from cell lines treated with 1 μM dCBP-1 PROTAC for the stated duration. An antibody against GFP was used to probe the eGFP tag on the Acyl-eCR constructs. The CBP_BRD.1x eCR runs at approximately 60 kDa, and the Empty-eGFP construct runs at 33 kDa. Non-specific bands are marked by an asterisk (*). Revert700 Total Protein Stain is used to show equal loading in lanes.
    Figure Legend Snippet: (A) Schematic showing how the Parbit expression cassette is used to generate stably expressed Acyl-eCRs in mESCs. RMCE by Cre, followed by a double selection of ganciclovir and puromycin, was applied to generate these constructs at a defined site in the mouse genome. A CAG promoter drives the constitutive expression of the bromodomain of interest, which is fused to a nuclear localization signal (NLS), and an eGFP tag. This construct also fuses a biotin acceptor site to the N-terminus of the protein, which can be biotinylated in vivo by a bacterial BirA ligase. (B) Schematic diagram showing how CBP was endogenously tagged with an eGFP tag. A homology donor construct was generated by cloning a 900 bp upstream and a 1,048 bp (CBP) or 1,068 bp (p300) downstream homology arm flanking a 30 bp flexible GGS linker that was fused to an eGFP tag. This donor construct was co-transfected with a pX330 CRISPR-Cas9 plasmid, which had an sgRNA targeting the C-terminus of the CBP gene. (C-D) Sanger sequencing of genotyping PCR products from the C-terminus of the CBP (C) and p300 (D) loci, confirming the in-frame homologous integration of an eGFP tag. Data shown are from mESC clone #1 for both CBP and p300 tagging. (E) Flow cytometry data showing the eGFP signal from cell lines where either CBP or p300 were endogenously tagged with eGFP. Two clonal replicates for each protein tagging are shown. Cell lines were maintained in culture for more than two weeks to demonstrate stable expression of the eGFP fusion proteins. (F) Western blot of nuclear extracts from cell lines treated with 1 μM dCBP-1 PROTAC for the stated duration. An antibody against GFP was used to probe the eGFP tag on the Acyl-eCR constructs. The CBP_BRD.1x eCR runs at approximately 60 kDa, and the Empty-eGFP construct runs at 33 kDa. Non-specific bands are marked by an asterisk (*). Revert700 Total Protein Stain is used to show equal loading in lanes.

    Techniques Used: Expressing, Stable Transfection, Selection, Construct, In Vivo, Generated, Cloning, Transfection, CRISPR, Plasmid Preparation, Sequencing, Flow Cytometry, Western Blot, Staining

    (A) Schematic showing the domain architecture of the Brd4 protein, and how its bromodomains are being used in several combinations to make acyl-eCRs and to determine how the valency of reader domains affects drug perturbations. (B) Immunofluorescence images of mESCs showing the nuclear localization of different valencies of the second bromodomain from BRD4 in the Parbit system (green) and their colocalization with Hoechst (magenta) after drug treatments. All scale bars are 5 µM. Drug treatments were performed at 1 μM concentrations for 24 hours. Bottom panel: Representative pseudocolored images (eGFP signal) depicting the differences in fluorescence intensities in different cell lines. A gradient pseudocolor bar (signal intensity) is shown at the left. (C) Normalized FACS data showing the effects of ARV-825 PROTAC treatment on cells expressing several combinations of bromodomains from BRD4. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.
    Figure Legend Snippet: (A) Schematic showing the domain architecture of the Brd4 protein, and how its bromodomains are being used in several combinations to make acyl-eCRs and to determine how the valency of reader domains affects drug perturbations. (B) Immunofluorescence images of mESCs showing the nuclear localization of different valencies of the second bromodomain from BRD4 in the Parbit system (green) and their colocalization with Hoechst (magenta) after drug treatments. All scale bars are 5 µM. Drug treatments were performed at 1 μM concentrations for 24 hours. Bottom panel: Representative pseudocolored images (eGFP signal) depicting the differences in fluorescence intensities in different cell lines. A gradient pseudocolor bar (signal intensity) is shown at the left. (C) Normalized FACS data showing the effects of ARV-825 PROTAC treatment on cells expressing several combinations of bromodomains from BRD4. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

    Techniques Used: Immunofluorescence, Fluorescence, Expressing

    (A) Top: Schematic showing how the competitive binding of small molecule inhibitors versus PROTACs for the binding pocket of Acyl-eCRs can be used to measure the affinity of a small molecule for a bromodomain in cellulo . Inhibitors with higher affinity for a bromodomain, better prevent PROTAC-induced degradation. Bottom : Treatment scheme for competitive binding experiments. Cells were treated with 1 μM inhibitors for 1 hour. Then, varying concentrations of the PROTAC were added in addition to the previously added inhibitor. After 3 hours of treatment, the cell fluorescence was measured via flow cytometry. (B) Competitive binding between ARV-825 and several small molecule inhibitors showing how the inhibitors bind to BRD4(2)_BRD.1x. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of ARV-825 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells. (C) Competitive binding between dCBP-1 and several small molecule inhibitors showing how the inhibitors bind CBP bromodomains in Acyl-eCR constructs versus the endogenous CBP protein. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of dCBP-1 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.
    Figure Legend Snippet: (A) Top: Schematic showing how the competitive binding of small molecule inhibitors versus PROTACs for the binding pocket of Acyl-eCRs can be used to measure the affinity of a small molecule for a bromodomain in cellulo . Inhibitors with higher affinity for a bromodomain, better prevent PROTAC-induced degradation. Bottom : Treatment scheme for competitive binding experiments. Cells were treated with 1 μM inhibitors for 1 hour. Then, varying concentrations of the PROTAC were added in addition to the previously added inhibitor. After 3 hours of treatment, the cell fluorescence was measured via flow cytometry. (B) Competitive binding between ARV-825 and several small molecule inhibitors showing how the inhibitors bind to BRD4(2)_BRD.1x. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of ARV-825 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells. (C) Competitive binding between dCBP-1 and several small molecule inhibitors showing how the inhibitors bind CBP bromodomains in Acyl-eCR constructs versus the endogenous CBP protein. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of dCBP-1 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

    Techniques Used: Binding Assay, Fluorescence, Flow Cytometry, Concentration Assay, Construct

    (A) Phylogenetic tree showing that the panel of Acyl-eCRs comprises representative bromodomains from all classes of bromodomains. All bromodomain protein sequences were obtained from InterPro and aligned with Clustal Omega’s multiple sequence alignment. Bromodomains are classified based on structure & druggability . Black circles represent the bromodomains that are included in the panel of Acyl-eCR cell lines. (B) Normalized FACS data showing the effects of dCBP-1 PROTAC treatments on all Acyl-eCR cell lines. PROTAC was added at a 1 μM concentration for 24 hours of treatment. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.
    Figure Legend Snippet: (A) Phylogenetic tree showing that the panel of Acyl-eCRs comprises representative bromodomains from all classes of bromodomains. All bromodomain protein sequences were obtained from InterPro and aligned with Clustal Omega’s multiple sequence alignment. Bromodomains are classified based on structure & druggability . Black circles represent the bromodomains that are included in the panel of Acyl-eCR cell lines. (B) Normalized FACS data showing the effects of dCBP-1 PROTAC treatments on all Acyl-eCR cell lines. PROTAC was added at a 1 μM concentration for 24 hours of treatment. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

    Techniques Used: Sequencing, Concentration Assay

    (A) Schematic showing how nuclei isolation & permeabilization followed by flow cytometry can measure the retention of proteins on chromatin in lieu of high background fluorescence. Nuclei can be harvested and permeabilized from whole cells, and then washed to remove the unbound or weakly bound fraction of the protein of interest. Since the weakly bound fraction of protein is removed, the fraction of protein remaining can be measured at a better signal-to-noise ratio via flow cytometry. (B) Flow cytometry analysis of nuclei harvested from BRD9_BRD.1x or WT cells. N3 gate shows the GFP signal being measured in nuclei, after iBRD9 or control treatments. Treatments in the WT cell line show a change in autofluorescence in the nuclei from the drug treatments. (C) Normalized flow cytometry data showing how Acyl-eCRs with 1x or 2x copies of the BRD9 bromodomain remain bound to chromatin, after iBRD9 treatments. iBRD9 treatments were performed at 1 μM concentration for 24 hours. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.
    Figure Legend Snippet: (A) Schematic showing how nuclei isolation & permeabilization followed by flow cytometry can measure the retention of proteins on chromatin in lieu of high background fluorescence. Nuclei can be harvested and permeabilized from whole cells, and then washed to remove the unbound or weakly bound fraction of the protein of interest. Since the weakly bound fraction of protein is removed, the fraction of protein remaining can be measured at a better signal-to-noise ratio via flow cytometry. (B) Flow cytometry analysis of nuclei harvested from BRD9_BRD.1x or WT cells. N3 gate shows the GFP signal being measured in nuclei, after iBRD9 or control treatments. Treatments in the WT cell line show a change in autofluorescence in the nuclei from the drug treatments. (C) Normalized flow cytometry data showing how Acyl-eCRs with 1x or 2x copies of the BRD9 bromodomain remain bound to chromatin, after iBRD9 treatments. iBRD9 treatments were performed at 1 μM concentration for 24 hours. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

    Techniques Used: Isolation, Flow Cytometry, Fluorescence, Control, Concentration Assay



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    Selleck Chemicals r0032 ibrd9 selleck
    Pharmaceutically targeting BRD9 enhances the antitumor effect of oHSV1 in vitro (A) Flow cytometry analysis of oHSV1-treated control or <t>IBRD9-pretreated</t> (1 μM, 24 h) CT2A Nectin1 and MGG4 cells subjected to PI/Annexin V staining for cell death analysis (PI+) ( n = 3). (B) Quantitative real-time PCR analysis of oHSV1 glycoprotein D levels in control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cells treated with oHSV1. GAPDH/Gapdh transcript normalization ( n = 3). (C) Plaque formation assay of oHSV1-treated control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cell culture medium. The virus titer was determined after 48 h ( n = 3). (D) Calreticulin (CRT) exposure analysis of control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 cells treated with oHSV1 ( n = 3). (E) Calreticulin (CRT) exposure analysis of control or IBRD9-pretreated (1 μM, 24 h) MGG4 cells treated with oHSV1 ( n = 3). (F) Extracellular ATP level analysis in control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cells treated with oHSV1 ( n = 3). (G) Extracellular HMGB1 level analysis in control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cells treated with oHSV1 ( n = 3). (H) In vitro co-culture proliferation experiments with OT-I CD8 + T cells and cDC1s generated from WT mice in the IBRD9- and oHSV1-treated CT2A Nectin1 -OVA-B2m −/− cells ( n = 3). (I) Schematic of human glioblastoma-derived organoid processing and verification of oHSV1-mediated killing by PI staining, 3D cell titer assays, and ICD marker analysis. (J) PI staining of IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1. Scale bars, 300 μm. (K) 3D cell viability assay of IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 ( n = 3). (L) Extracellular ATP-level analysis in control or IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 ( n = 3). (M) Extracellular HMGB1-level analysis in control or IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 ( n = 3). (N) Schematic of human glioblastoma-derived tumor slice processing and verification of oHSV1 replication by anti-HSV1 staining and quantitative real-time PCR. (O) Representative immunohistochemistry images of HSV1 staining in IBRD9-pretreated or control human glioblastoma-derived tumor slices. Scale bars, 50 μm. (P) Analysis of oHSV1 replication in IBRD9-pretreated (2 μM, 24 h) or control human glioblastoma-derived tumor slices. After oHSV1 treatment, the distribution of oHSV1 in the sections was detected by anti-HSV1 immunohistochemistry ( n = 3). (Q) Quantitative real-time PCR analysis of oHSV1 glycoprotein D levels in control or IBRD9-pretreated (2 μM, 24 h) human glioblastoma-derived tumor sections treated with oHSV1. GAPDH transcript normalization ( n = 3). Data represent mean ± SD. Two-way ANOVA (A, D, E, F, G, H, K, L, and M), unpaired two-tailed Student’s t test (B, C, P, and Q). The diagrams (I and N) were created using BioRender. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant.
    R0032 Ibrd9 Selleck, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    s7835  (Selleck Chemicals)
    93
    Selleck Chemicals s7835
    Pharmaceutically targeting BRD9 enhances the antitumor effect of oHSV1 in vitro (A) Flow cytometry analysis of oHSV1-treated control or <t>IBRD9-pretreated</t> (1 μM, 24 h) CT2A Nectin1 and MGG4 cells subjected to PI/Annexin V staining for cell death analysis (PI+) ( n = 3). (B) Quantitative real-time PCR analysis of oHSV1 glycoprotein D levels in control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cells treated with oHSV1. GAPDH/Gapdh transcript normalization ( n = 3). (C) Plaque formation assay of oHSV1-treated control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cell culture medium. The virus titer was determined after 48 h ( n = 3). (D) Calreticulin (CRT) exposure analysis of control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 cells treated with oHSV1 ( n = 3). (E) Calreticulin (CRT) exposure analysis of control or IBRD9-pretreated (1 μM, 24 h) MGG4 cells treated with oHSV1 ( n = 3). (F) Extracellular ATP level analysis in control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cells treated with oHSV1 ( n = 3). (G) Extracellular HMGB1 level analysis in control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cells treated with oHSV1 ( n = 3). (H) In vitro co-culture proliferation experiments with OT-I CD8 + T cells and cDC1s generated from WT mice in the IBRD9- and oHSV1-treated CT2A Nectin1 -OVA-B2m −/− cells ( n = 3). (I) Schematic of human glioblastoma-derived organoid processing and verification of oHSV1-mediated killing by PI staining, 3D cell titer assays, and ICD marker analysis. (J) PI staining of IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1. Scale bars, 300 μm. (K) 3D cell viability assay of IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 ( n = 3). (L) Extracellular ATP-level analysis in control or IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 ( n = 3). (M) Extracellular HMGB1-level analysis in control or IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 ( n = 3). (N) Schematic of human glioblastoma-derived tumor slice processing and verification of oHSV1 replication by anti-HSV1 staining and quantitative real-time PCR. (O) Representative immunohistochemistry images of HSV1 staining in IBRD9-pretreated or control human glioblastoma-derived tumor slices. Scale bars, 50 μm. (P) Analysis of oHSV1 replication in IBRD9-pretreated (2 μM, 24 h) or control human glioblastoma-derived tumor slices. After oHSV1 treatment, the distribution of oHSV1 in the sections was detected by anti-HSV1 immunohistochemistry ( n = 3). (Q) Quantitative real-time PCR analysis of oHSV1 glycoprotein D levels in control or IBRD9-pretreated (2 μM, 24 h) human glioblastoma-derived tumor sections treated with oHSV1. GAPDH transcript normalization ( n = 3). Data represent mean ± SD. Two-way ANOVA (A, D, E, F, G, H, K, L, and M), unpaired two-tailed Student’s t test (B, C, P, and Q). The diagrams (I and N) were created using BioRender. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant.
    S7835, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
    s7835 - by Bioz Stars, 2026-05
    93/100 stars
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    (A) Schematic showing how the Parbit expression cassette is used to generate stably expressed Acyl-eCRs in mESCs. RMCE by Cre, followed by a double selection of ganciclovir and puromycin, was applied to generate these constructs at a defined site in the mouse genome. A CAG promoter drives the constitutive expression of the bromodomain of interest, which is fused to a nuclear localization signal (NLS), and an eGFP tag. This construct also fuses a biotin acceptor site to the N-terminus of the protein, which can be biotinylated in vivo by a bacterial BirA ligase. (B) Schematic diagram showing how CBP was endogenously tagged with an eGFP tag. A homology donor construct was generated by cloning a 900 bp upstream and a 1,048 bp (CBP) or 1,068 bp (p300) downstream homology arm flanking a 30 bp flexible GGS linker that was fused to an eGFP tag. This donor construct was co-transfected with a pX330 CRISPR-Cas9 plasmid, which had an sgRNA targeting the C-terminus of the CBP gene. (C-D) Sanger sequencing of genotyping PCR products from the C-terminus of the CBP (C) and p300 (D) loci, confirming the in-frame homologous integration of an eGFP tag. Data shown are from mESC clone #1 for both CBP and p300 tagging. (E) Flow cytometry data showing the eGFP signal from cell lines where either CBP or p300 were endogenously tagged with eGFP. Two clonal replicates for each protein tagging are shown. Cell lines were maintained in culture for more than two weeks to demonstrate stable expression of the eGFP fusion proteins. (F) Western blot of nuclear extracts from cell lines treated with 1 μM dCBP-1 PROTAC for the stated duration. An antibody against GFP was used to probe the eGFP tag on the Acyl-eCR constructs. The CBP_BRD.1x eCR runs at approximately 60 kDa, and the Empty-eGFP construct runs at 33 kDa. Non-specific bands are marked by an asterisk (*). Revert700 Total Protein Stain is used to show equal loading in lanes.

    Journal: bioRxiv

    Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

    doi: 10.1101/2025.09.06.674632

    Figure Lengend Snippet: (A) Schematic showing how the Parbit expression cassette is used to generate stably expressed Acyl-eCRs in mESCs. RMCE by Cre, followed by a double selection of ganciclovir and puromycin, was applied to generate these constructs at a defined site in the mouse genome. A CAG promoter drives the constitutive expression of the bromodomain of interest, which is fused to a nuclear localization signal (NLS), and an eGFP tag. This construct also fuses a biotin acceptor site to the N-terminus of the protein, which can be biotinylated in vivo by a bacterial BirA ligase. (B) Schematic diagram showing how CBP was endogenously tagged with an eGFP tag. A homology donor construct was generated by cloning a 900 bp upstream and a 1,048 bp (CBP) or 1,068 bp (p300) downstream homology arm flanking a 30 bp flexible GGS linker that was fused to an eGFP tag. This donor construct was co-transfected with a pX330 CRISPR-Cas9 plasmid, which had an sgRNA targeting the C-terminus of the CBP gene. (C-D) Sanger sequencing of genotyping PCR products from the C-terminus of the CBP (C) and p300 (D) loci, confirming the in-frame homologous integration of an eGFP tag. Data shown are from mESC clone #1 for both CBP and p300 tagging. (E) Flow cytometry data showing the eGFP signal from cell lines where either CBP or p300 were endogenously tagged with eGFP. Two clonal replicates for each protein tagging are shown. Cell lines were maintained in culture for more than two weeks to demonstrate stable expression of the eGFP fusion proteins. (F) Western blot of nuclear extracts from cell lines treated with 1 μM dCBP-1 PROTAC for the stated duration. An antibody against GFP was used to probe the eGFP tag on the Acyl-eCR constructs. The CBP_BRD.1x eCR runs at approximately 60 kDa, and the Empty-eGFP construct runs at 33 kDa. Non-specific bands are marked by an asterisk (*). Revert700 Total Protein Stain is used to show equal loading in lanes.

    Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

    Techniques: Expressing, Stable Transfection, Selection, Construct, In Vivo, Generated, Cloning, Transfection, CRISPR, Plasmid Preparation, Sequencing, Flow Cytometry, Western Blot, Staining

    (A) Schematic showing the domain architecture of the Brd4 protein, and how its bromodomains are being used in several combinations to make acyl-eCRs and to determine how the valency of reader domains affects drug perturbations. (B) Immunofluorescence images of mESCs showing the nuclear localization of different valencies of the second bromodomain from BRD4 in the Parbit system (green) and their colocalization with Hoechst (magenta) after drug treatments. All scale bars are 5 µM. Drug treatments were performed at 1 μM concentrations for 24 hours. Bottom panel: Representative pseudocolored images (eGFP signal) depicting the differences in fluorescence intensities in different cell lines. A gradient pseudocolor bar (signal intensity) is shown at the left. (C) Normalized FACS data showing the effects of ARV-825 PROTAC treatment on cells expressing several combinations of bromodomains from BRD4. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

    Journal: bioRxiv

    Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

    doi: 10.1101/2025.09.06.674632

    Figure Lengend Snippet: (A) Schematic showing the domain architecture of the Brd4 protein, and how its bromodomains are being used in several combinations to make acyl-eCRs and to determine how the valency of reader domains affects drug perturbations. (B) Immunofluorescence images of mESCs showing the nuclear localization of different valencies of the second bromodomain from BRD4 in the Parbit system (green) and their colocalization with Hoechst (magenta) after drug treatments. All scale bars are 5 µM. Drug treatments were performed at 1 μM concentrations for 24 hours. Bottom panel: Representative pseudocolored images (eGFP signal) depicting the differences in fluorescence intensities in different cell lines. A gradient pseudocolor bar (signal intensity) is shown at the left. (C) Normalized FACS data showing the effects of ARV-825 PROTAC treatment on cells expressing several combinations of bromodomains from BRD4. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

    Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

    Techniques: Immunofluorescence, Fluorescence, Expressing

    (A) Top: Schematic showing how the competitive binding of small molecule inhibitors versus PROTACs for the binding pocket of Acyl-eCRs can be used to measure the affinity of a small molecule for a bromodomain in cellulo . Inhibitors with higher affinity for a bromodomain, better prevent PROTAC-induced degradation. Bottom : Treatment scheme for competitive binding experiments. Cells were treated with 1 μM inhibitors for 1 hour. Then, varying concentrations of the PROTAC were added in addition to the previously added inhibitor. After 3 hours of treatment, the cell fluorescence was measured via flow cytometry. (B) Competitive binding between ARV-825 and several small molecule inhibitors showing how the inhibitors bind to BRD4(2)_BRD.1x. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of ARV-825 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells. (C) Competitive binding between dCBP-1 and several small molecule inhibitors showing how the inhibitors bind CBP bromodomains in Acyl-eCR constructs versus the endogenous CBP protein. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of dCBP-1 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

    Journal: bioRxiv

    Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

    doi: 10.1101/2025.09.06.674632

    Figure Lengend Snippet: (A) Top: Schematic showing how the competitive binding of small molecule inhibitors versus PROTACs for the binding pocket of Acyl-eCRs can be used to measure the affinity of a small molecule for a bromodomain in cellulo . Inhibitors with higher affinity for a bromodomain, better prevent PROTAC-induced degradation. Bottom : Treatment scheme for competitive binding experiments. Cells were treated with 1 μM inhibitors for 1 hour. Then, varying concentrations of the PROTAC were added in addition to the previously added inhibitor. After 3 hours of treatment, the cell fluorescence was measured via flow cytometry. (B) Competitive binding between ARV-825 and several small molecule inhibitors showing how the inhibitors bind to BRD4(2)_BRD.1x. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of ARV-825 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells. (C) Competitive binding between dCBP-1 and several small molecule inhibitors showing how the inhibitors bind CBP bromodomains in Acyl-eCR constructs versus the endogenous CBP protein. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of dCBP-1 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

    Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

    Techniques: Binding Assay, Fluorescence, Flow Cytometry, Concentration Assay, Construct

    (A) Phylogenetic tree showing that the panel of Acyl-eCRs comprises representative bromodomains from all classes of bromodomains. All bromodomain protein sequences were obtained from InterPro and aligned with Clustal Omega’s multiple sequence alignment. Bromodomains are classified based on structure & druggability . Black circles represent the bromodomains that are included in the panel of Acyl-eCR cell lines. (B) Normalized FACS data showing the effects of dCBP-1 PROTAC treatments on all Acyl-eCR cell lines. PROTAC was added at a 1 μM concentration for 24 hours of treatment. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

    Journal: bioRxiv

    Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

    doi: 10.1101/2025.09.06.674632

    Figure Lengend Snippet: (A) Phylogenetic tree showing that the panel of Acyl-eCRs comprises representative bromodomains from all classes of bromodomains. All bromodomain protein sequences were obtained from InterPro and aligned with Clustal Omega’s multiple sequence alignment. Bromodomains are classified based on structure & druggability . Black circles represent the bromodomains that are included in the panel of Acyl-eCR cell lines. (B) Normalized FACS data showing the effects of dCBP-1 PROTAC treatments on all Acyl-eCR cell lines. PROTAC was added at a 1 μM concentration for 24 hours of treatment. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

    Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

    Techniques: Sequencing, Concentration Assay

    (A) Schematic showing how nuclei isolation & permeabilization followed by flow cytometry can measure the retention of proteins on chromatin in lieu of high background fluorescence. Nuclei can be harvested and permeabilized from whole cells, and then washed to remove the unbound or weakly bound fraction of the protein of interest. Since the weakly bound fraction of protein is removed, the fraction of protein remaining can be measured at a better signal-to-noise ratio via flow cytometry. (B) Flow cytometry analysis of nuclei harvested from BRD9_BRD.1x or WT cells. N3 gate shows the GFP signal being measured in nuclei, after iBRD9 or control treatments. Treatments in the WT cell line show a change in autofluorescence in the nuclei from the drug treatments. (C) Normalized flow cytometry data showing how Acyl-eCRs with 1x or 2x copies of the BRD9 bromodomain remain bound to chromatin, after iBRD9 treatments. iBRD9 treatments were performed at 1 μM concentration for 24 hours. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

    Journal: bioRxiv

    Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

    doi: 10.1101/2025.09.06.674632

    Figure Lengend Snippet: (A) Schematic showing how nuclei isolation & permeabilization followed by flow cytometry can measure the retention of proteins on chromatin in lieu of high background fluorescence. Nuclei can be harvested and permeabilized from whole cells, and then washed to remove the unbound or weakly bound fraction of the protein of interest. Since the weakly bound fraction of protein is removed, the fraction of protein remaining can be measured at a better signal-to-noise ratio via flow cytometry. (B) Flow cytometry analysis of nuclei harvested from BRD9_BRD.1x or WT cells. N3 gate shows the GFP signal being measured in nuclei, after iBRD9 or control treatments. Treatments in the WT cell line show a change in autofluorescence in the nuclei from the drug treatments. (C) Normalized flow cytometry data showing how Acyl-eCRs with 1x or 2x copies of the BRD9 bromodomain remain bound to chromatin, after iBRD9 treatments. iBRD9 treatments were performed at 1 μM concentration for 24 hours. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

    Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

    Techniques: Isolation, Flow Cytometry, Fluorescence, Control, Concentration Assay

    (A) Schematic showing how nuclei isolation & permeabilization followed by flow cytometry can measure the retention of proteins on chromatin in lieu of high background fluorescence. Nuclei can be harvested and permeabilized from whole cells, and then washed to remove the unbound or weakly bound fraction of the protein of interest. Since the weakly bound fraction of protein is removed, the fraction of protein remaining can be measured at a better signal-to-noise ratio via flow cytometry. (B) Flow cytometry analysis of nuclei harvested from BRD9_BRD.1x or WT cells. N3 gate shows the GFP signal being measured in nuclei, after iBRD9 or control treatments. Treatments in the WT cell line show a change in autofluorescence in the nuclei from the drug treatments. (C) Normalized flow cytometry data showing how Acyl-eCRs with 1x or 2x copies of the BRD9 bromodomain remain bound to chromatin, after iBRD9 treatments. iBRD9 treatments were performed at 1 μM concentration for 24 hours. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

    Journal: bioRxiv

    Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

    doi: 10.1101/2025.09.06.674632

    Figure Lengend Snippet: (A) Schematic showing how nuclei isolation & permeabilization followed by flow cytometry can measure the retention of proteins on chromatin in lieu of high background fluorescence. Nuclei can be harvested and permeabilized from whole cells, and then washed to remove the unbound or weakly bound fraction of the protein of interest. Since the weakly bound fraction of protein is removed, the fraction of protein remaining can be measured at a better signal-to-noise ratio via flow cytometry. (B) Flow cytometry analysis of nuclei harvested from BRD9_BRD.1x or WT cells. N3 gate shows the GFP signal being measured in nuclei, after iBRD9 or control treatments. Treatments in the WT cell line show a change in autofluorescence in the nuclei from the drug treatments. (C) Normalized flow cytometry data showing how Acyl-eCRs with 1x or 2x copies of the BRD9 bromodomain remain bound to chromatin, after iBRD9 treatments. iBRD9 treatments were performed at 1 μM concentration for 24 hours. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

    Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

    Techniques: Isolation, Flow Cytometry, Fluorescence, Control, Concentration Assay

    Pharmaceutically targeting BRD9 enhances the antitumor effect of oHSV1 in vitro (A) Flow cytometry analysis of oHSV1-treated control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cells subjected to PI/Annexin V staining for cell death analysis (PI+) ( n = 3). (B) Quantitative real-time PCR analysis of oHSV1 glycoprotein D levels in control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cells treated with oHSV1. GAPDH/Gapdh transcript normalization ( n = 3). (C) Plaque formation assay of oHSV1-treated control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cell culture medium. The virus titer was determined after 48 h ( n = 3). (D) Calreticulin (CRT) exposure analysis of control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 cells treated with oHSV1 ( n = 3). (E) Calreticulin (CRT) exposure analysis of control or IBRD9-pretreated (1 μM, 24 h) MGG4 cells treated with oHSV1 ( n = 3). (F) Extracellular ATP level analysis in control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cells treated with oHSV1 ( n = 3). (G) Extracellular HMGB1 level analysis in control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cells treated with oHSV1 ( n = 3). (H) In vitro co-culture proliferation experiments with OT-I CD8 + T cells and cDC1s generated from WT mice in the IBRD9- and oHSV1-treated CT2A Nectin1 -OVA-B2m −/− cells ( n = 3). (I) Schematic of human glioblastoma-derived organoid processing and verification of oHSV1-mediated killing by PI staining, 3D cell titer assays, and ICD marker analysis. (J) PI staining of IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1. Scale bars, 300 μm. (K) 3D cell viability assay of IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 ( n = 3). (L) Extracellular ATP-level analysis in control or IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 ( n = 3). (M) Extracellular HMGB1-level analysis in control or IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 ( n = 3). (N) Schematic of human glioblastoma-derived tumor slice processing and verification of oHSV1 replication by anti-HSV1 staining and quantitative real-time PCR. (O) Representative immunohistochemistry images of HSV1 staining in IBRD9-pretreated or control human glioblastoma-derived tumor slices. Scale bars, 50 μm. (P) Analysis of oHSV1 replication in IBRD9-pretreated (2 μM, 24 h) or control human glioblastoma-derived tumor slices. After oHSV1 treatment, the distribution of oHSV1 in the sections was detected by anti-HSV1 immunohistochemistry ( n = 3). (Q) Quantitative real-time PCR analysis of oHSV1 glycoprotein D levels in control or IBRD9-pretreated (2 μM, 24 h) human glioblastoma-derived tumor sections treated with oHSV1. GAPDH transcript normalization ( n = 3). Data represent mean ± SD. Two-way ANOVA (A, D, E, F, G, H, K, L, and M), unpaired two-tailed Student’s t test (B, C, P, and Q). The diagrams (I and N) were created using BioRender. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant.

    Journal: Cell Reports Medicine

    Article Title: BRD9 inhibition overcomes oncolytic virus therapy resistance in glioblastoma

    doi: 10.1016/j.xcrm.2025.102258

    Figure Lengend Snippet: Pharmaceutically targeting BRD9 enhances the antitumor effect of oHSV1 in vitro (A) Flow cytometry analysis of oHSV1-treated control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cells subjected to PI/Annexin V staining for cell death analysis (PI+) ( n = 3). (B) Quantitative real-time PCR analysis of oHSV1 glycoprotein D levels in control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cells treated with oHSV1. GAPDH/Gapdh transcript normalization ( n = 3). (C) Plaque formation assay of oHSV1-treated control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cell culture medium. The virus titer was determined after 48 h ( n = 3). (D) Calreticulin (CRT) exposure analysis of control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 cells treated with oHSV1 ( n = 3). (E) Calreticulin (CRT) exposure analysis of control or IBRD9-pretreated (1 μM, 24 h) MGG4 cells treated with oHSV1 ( n = 3). (F) Extracellular ATP level analysis in control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cells treated with oHSV1 ( n = 3). (G) Extracellular HMGB1 level analysis in control or IBRD9-pretreated (1 μM, 24 h) CT2A Nectin1 and MGG4 cells treated with oHSV1 ( n = 3). (H) In vitro co-culture proliferation experiments with OT-I CD8 + T cells and cDC1s generated from WT mice in the IBRD9- and oHSV1-treated CT2A Nectin1 -OVA-B2m −/− cells ( n = 3). (I) Schematic of human glioblastoma-derived organoid processing and verification of oHSV1-mediated killing by PI staining, 3D cell titer assays, and ICD marker analysis. (J) PI staining of IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1. Scale bars, 300 μm. (K) 3D cell viability assay of IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 ( n = 3). (L) Extracellular ATP-level analysis in control or IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 ( n = 3). (M) Extracellular HMGB1-level analysis in control or IBRD9-pretreated (1 μM, 24 h) human glioblastoma-derived organoids treated with oHSV1 ( n = 3). (N) Schematic of human glioblastoma-derived tumor slice processing and verification of oHSV1 replication by anti-HSV1 staining and quantitative real-time PCR. (O) Representative immunohistochemistry images of HSV1 staining in IBRD9-pretreated or control human glioblastoma-derived tumor slices. Scale bars, 50 μm. (P) Analysis of oHSV1 replication in IBRD9-pretreated (2 μM, 24 h) or control human glioblastoma-derived tumor slices. After oHSV1 treatment, the distribution of oHSV1 in the sections was detected by anti-HSV1 immunohistochemistry ( n = 3). (Q) Quantitative real-time PCR analysis of oHSV1 glycoprotein D levels in control or IBRD9-pretreated (2 μM, 24 h) human glioblastoma-derived tumor sections treated with oHSV1. GAPDH transcript normalization ( n = 3). Data represent mean ± SD. Two-way ANOVA (A, D, E, F, G, H, K, L, and M), unpaired two-tailed Student’s t test (B, C, P, and Q). The diagrams (I and N) were created using BioRender. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant.

    Article Snippet: IBRD9 , Selleck , Cat# S7835.

    Techniques: In Vitro, Flow Cytometry, Control, Staining, Real-time Polymerase Chain Reaction, Plaque Formation Assay, Cell Culture, Virus, Co-Culture Assay, Generated, Derivative Assay, Marker, Viability Assay, Immunohistochemistry, Two Tailed Test

    Pharmaceutically targeting BRD9 enhances the antitumor effect of oHSV1 in vivo , and BRD9 expression is associated with poor clinical outcome in cancer patients treated with oHSV1 (A) Survival curve of IBRD9, oHSV1, and ICB (the anti-mouse PD-1 antibody and the anti-mouse CTLA4 antibody) combination therapy in CT2A Nectin1 tumor-bearing mice ( n = 5). (B) Survival curve of IBRD9, oHSV1, and ICB (the anti-mouse PD-1 antibody and the anti-mouse CTLA4 antibody) combination therapy in GL261 Nectin1 tumor-bearing mice ( n = 5). (C) Survival curve of long-term survivor mice and age-matched control mice challenged with CT2A Nectin1 cells ( n = 5). (D) Survival curve of long-term survivor mice and age-matched control mice challenged with GL261 Nectin1 cells ( n = 5). (E) Kaplan-Meier analysis showing the PFS of patients with glioblastoma treated with oHSV1 subdivided by the expression of BRD9 (high-expression patients: n = 7; low-expression patients: n = 6). HR, hazard ratio. (F) Analysis of BRD9 expression in liver cancer and pancreatic cancer biopsy sections from patients participating in an oHSV1 clinical trial. Representative immunohistochemistry images of BRD9 staining (oHSV1 response: SD, stable disease; oHSV1 nonresponse: PD, progressive disease). Scale bars, 50 μm. (G) BRD9-stained sections were quantified by H-score (SD: n = 14; PD: n = 13). Data represent mean ± SD. Unpaired two-tailed Student’s t test (G) and log rank test (A–E). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant.

    Journal: Cell Reports Medicine

    Article Title: BRD9 inhibition overcomes oncolytic virus therapy resistance in glioblastoma

    doi: 10.1016/j.xcrm.2025.102258

    Figure Lengend Snippet: Pharmaceutically targeting BRD9 enhances the antitumor effect of oHSV1 in vivo , and BRD9 expression is associated with poor clinical outcome in cancer patients treated with oHSV1 (A) Survival curve of IBRD9, oHSV1, and ICB (the anti-mouse PD-1 antibody and the anti-mouse CTLA4 antibody) combination therapy in CT2A Nectin1 tumor-bearing mice ( n = 5). (B) Survival curve of IBRD9, oHSV1, and ICB (the anti-mouse PD-1 antibody and the anti-mouse CTLA4 antibody) combination therapy in GL261 Nectin1 tumor-bearing mice ( n = 5). (C) Survival curve of long-term survivor mice and age-matched control mice challenged with CT2A Nectin1 cells ( n = 5). (D) Survival curve of long-term survivor mice and age-matched control mice challenged with GL261 Nectin1 cells ( n = 5). (E) Kaplan-Meier analysis showing the PFS of patients with glioblastoma treated with oHSV1 subdivided by the expression of BRD9 (high-expression patients: n = 7; low-expression patients: n = 6). HR, hazard ratio. (F) Analysis of BRD9 expression in liver cancer and pancreatic cancer biopsy sections from patients participating in an oHSV1 clinical trial. Representative immunohistochemistry images of BRD9 staining (oHSV1 response: SD, stable disease; oHSV1 nonresponse: PD, progressive disease). Scale bars, 50 μm. (G) BRD9-stained sections were quantified by H-score (SD: n = 14; PD: n = 13). Data represent mean ± SD. Unpaired two-tailed Student’s t test (G) and log rank test (A–E). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant.

    Article Snippet: IBRD9 , Selleck , Cat# S7835.

    Techniques: In Vivo, Expressing, Control, Immunohistochemistry, Staining, Two Tailed Test