Journal: Nature Communications

Article Title: BRD9-mediated chromatin remodeling suppresses osteoclastogenesis through negative feedback mechanism

doi: 10.1038/s41467-023-37116-5

Figure Lengend Snippet: a TRAP staining and quantification analysis of BMDMs from 4-week-old LysM-Cre;Brd9 fl/fl mice and littermate control mice after 3 days of RANKL-induction. Scale bar, 200 μm. n = 8. b The mRNA expression of osteoclastic-specific genes of Acp5 , Ctsk, and Mmp9 in BMDMs from LysM-Cre;Brd9 fl/fl mice compared with that from control littermates after 3 days of RANKL-induction, as measured by qPCR. n = 3 for LysM-Cre;Brd9 fl/fl mice group. n = 6 for control littermates. c The protein expression of MMP9 and CTSK in BMDMs from LysM-Cre;Brd9 fl/fl mice compared with that from control littermates after 3 days of RANKL-induction, as measured by western blot. d Cell viability of RANKL-induced BMDMs with 1 day of iBRD9 at different concentration, shown by cell counting kit 8 assay. n = 10 biologically independent samples. e The mRNA expression of Acp5 and Mmp9 in RANKL-induced BMDMs with 1 day of iBRD9 at different concentration, as measured by qPCR. n = 5 biologically independent samples for Acp5 . n = 3 biologically independent samples for Mmp9 . f TRAP staining and quantification analysis of RANKL-induced BMDMs with 5 days of iBRD9 at different concentration. Scale bar, 200 μm. n = 5 biologically independent samples. g The protein expression of FOS and CTSK in BMDMs after 3 days of 1 μM iBRD9 or vector treatment with or without RANKL-induction, as measured by western blot. M-CSF, M; RANKL, R. h The mRNA expression of Acp5 and Mmp9 in BMDMs after 3 days of 1 μM iBRD9 or vector treatment with or without RANKL-induction, as measured by qPCR. M-CSF, M; RANKL, R. n = 3 biologically independent samples. All data in this figure are represented as mean ± SD. Two-tailed Student’s t -test for ( a ) and ( b ). One-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test for ( d ), ( e ), and ( f ); ANOVA with Tukey’s multiple comparisons test for ( h ). All experiments were performed in triplicates unless otherwise stated. Source data are provided in the Source data file.

Article Snippet: For the BRD9 inhibitor/degrader experiments, the cells were treated with either vehicle or varying concentrations of iBRD9 (Tocris Bioscience, 5591) or dBRD9 (R&D Systems, 6606) twenty-four hours after plated, as well as IFN-β1 cytokine (0.0625 ng/ml, R&D Systems, 8234-MB), DEX (10 −11 −10 −9 M), JQ1 (0.25 μM, Abmole Bioscience, M2167) and ZOL (10 μM, Abmole Bioscience, M5032).

Techniques: Staining, Control, Expressing, Western Blot, Concentration Assay, Cell Counting, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: BRD9-mediated chromatin remodeling suppresses osteoclastogenesis through negative feedback mechanism

doi: 10.1038/s41467-023-37116-5

Figure Lengend Snippet: a Heatmap hierarchical clustering and the MA plot showing the gene expression profiles in BMDMs after 1 day of BRD9 inhibition during osteoclastic differentiation with M-CSF and RANKL (MR). n = 4. Color scale represents normalized gene FPKM value by z-score scheme. b The top 10 Gene ontology (GO) biological process enriched in the comparison between BMDMs in MR + vector and MR + iBRD9 group . c Gene set enrichment analysis (GSEA) analysis of the top downregulated gene sets in MR + iBRD9 group compared with control group. d GSEA analysis of the top upregulated gene sets in MR + iBRD9 group compared with control group. e The protein expression of IFN-β, STAT2, and STAT1 in BMDMs at different days after osteoclastic differentiation, as measured by western blot. f GSEA plots and heatmap hierarchical clustering of GO term_0035458 in MR + iBRD9 group and control group. n = 4. g The mRNA expression of Acp5 and Mmp9 in BMDMs treated with 2 days of 1 μM iBRD9 and 0.0625 ng/ml IFN-β1 during osteoclastic induction, as measured by qPCR. n = 3 biologically independent samples. h TRAP staining of BMDMs treated with 3 days of 1 μM iBRD9 and 0.0625 ng/ml IFN-β1 during osteoclastic induction. Scale bar, 200 μm. All data in this figure are represented as mean ± SD. Hypergeometric distribution test for ( b ). Empirical phenotype-based permutation test for ( c ) and ( d ). One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test for ( g ). All experiments were performed in triplicates unless otherwise stated. Source data are provided in the Source data file.

Article Snippet: For the BRD9 inhibitor/degrader experiments, the cells were treated with either vehicle or varying concentrations of iBRD9 (Tocris Bioscience, 5591) or dBRD9 (R&D Systems, 6606) twenty-four hours after plated, as well as IFN-β1 cytokine (0.0625 ng/ml, R&D Systems, 8234-MB), DEX (10 −11 −10 −9 M), JQ1 (0.25 μM, Abmole Bioscience, M2167) and ZOL (10 μM, Abmole Bioscience, M5032).

Techniques: Gene Expression, Inhibition, Comparison, Plasmid Preparation, Control, Expressing, Western Blot, Staining

Journal: Nature Communications

Article Title: BRD9-mediated chromatin remodeling suppresses osteoclastogenesis through negative feedback mechanism

doi: 10.1038/s41467-023-37116-5

Figure Lengend Snippet: a Venn diagram showing overlapping genes, where BRD9 binding, RANKL-induced while downregulated after BRD9 inhibition. b KEGG pathway annotation of the genes, where BRD9 binding, RANKL-induced while downregulated after BRD9 inhibition. c Protein-protein interaction (PPI) networks of the genes, where BRD9 binding, RANKL-induced while downregulated after BRD9 inhibition. d Immunofluorescence staining of STAT1 (red) and CTSK (green) in the mouse distal femur at 4 weeks of age. Scale bar,100 μm. e Immunofluorescence of STAT1 (green), TRAP staining (red) and actin (red) in BMDMs after 3 days of RANKL-induction. Scale bar, 100 μm. f The mRNA expression of Stat1 in BMDMs from LysM-Cre;Brd9 fl/fl mice compared with that from control littermates after 3 days of RANKL-induction. n = 3 for LysM-Cre;Brd9 fl/fl mice group. n = 6 for control littermates. g The mRNA expression of Stat1 in BMDMs treated with iBRD9 after 2 days of RANKL-induction. n = 3 biologically independent samples. h The protein expression of STAT1 in BMDMs treated with iBRD9 after 2 days of RANKL-induction. i The protein expression of STAT1 in RAW264.7 transfected with control lentivirus (NC) and Stat1 -overexpression lentivirus (OE-Stat1). j The mRNA expression of Mmp9 , Acp5 and Fos in Stat1 -overexpressed and control RAW264.7 treated with iBRD9 after 2 days of RANKL-induction. n = 4 biologically independent samples. k Luciferase reporter activities of the Stat1 promoter alone or in the presence of enhancer region in osteoclastic induced RAW264.7 treated with iBRD9 or control vector. n = 5 biologically independent samples. l ChIP assay with BRD9 antibody (or IgG) in RAW264.7 cell line during osteoclastic induction. n = 3 biologically independent samples. All data in this figure are represented as mean ± SD. Two-tailed Student’s t -test for ( f ), ( j ), ( k ), and ( l ). One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test for ( g ). M, M-CSF; MR, M-CSF + RANKL. All experiments were performed in triplicates unless otherwise stated. Source data are provided in the Source data file.

Article Snippet: For the BRD9 inhibitor/degrader experiments, the cells were treated with either vehicle or varying concentrations of iBRD9 (Tocris Bioscience, 5591) or dBRD9 (R&D Systems, 6606) twenty-four hours after plated, as well as IFN-β1 cytokine (0.0625 ng/ml, R&D Systems, 8234-MB), DEX (10 −11 −10 −9 M), JQ1 (0.25 μM, Abmole Bioscience, M2167) and ZOL (10 μM, Abmole Bioscience, M5032).

Techniques: Binding Assay, Inhibition, Immunofluorescence, Staining, Expressing, Control, Transfection, Over Expression, Luciferase, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: BRD9-mediated chromatin remodeling suppresses osteoclastogenesis through negative feedback mechanism

doi: 10.1038/s41467-023-37116-5

Figure Lengend Snippet: a Heatmap showing the average ATAC-Seq signal centered on the transcription start site (TSS) of the nearest genes in osteoclastic induced BMDMs after control and iBRD9 treatment. Color scale represents average signal intensity. b Venn diagram showing the overlapping and discrepant peaks between control and iBRD9 group. c Volcano plot depicting differentially accessible region (DAR) between control and iBRD9 group. d GO terms of genes with gain and loss DAR around TSS after BRD9 inhibition. e Dot bubble plot showing the loss DAR motif enrichment after BRD9 inhibition. f Motifs enriched in BRD9 binding while loss after BRD9 inhibition. g The FOXP1 motif logo. h FOXP1 immunofluorescence (red) and CTSK (green) in the mouse distal femur at 4 weeks. Scale bar, 100 μm. i FOXP1 immunofluorescence (green), TRAP staining (red) and actin (red) in BMDMs after 3 days of RANKL-induction. Scale bar, 100 μm. j Co-IP assay with FOXP1 antibody (or IgG) in BMDMs during osteoclastic induction, followed by immunoblotting of BRD9 and FOXP1. k ChIP assay with FOXP1 antibody (or IgG) in RAW264.7 cell during RANKL-induction treated with iBRD9 or vector. n = 3 biologically independent samples. l The mRNA of Foxp1 , Stat1 , Myc , Mmp9 and Acp5 in osteoclastic induced RAW264.7 cell treated with Foxp1 knockdown lentivirus ( Foxp1 -Lenti) or control lentivirus (Control-Lenti). n = 3 biologically independent samples. m The mRNA of Foxp1 in BMDMs from LysM-Cre;Brd9 fl/fl ( n = 3) and control mice ( n = 6) at 4 weeks after 3 days of osteoclast differentiation. n The mRNA of Foxp1 in BMDMs treated with iBRD9 after 3 days of osteoclast differentiation. n = 3 biologically independent samples. o Schematic drawing shows chromatin remodeling mediated by BRD9. All data in this figure are represented as mean ± SD. Negative binomial distribution used for c . Hypergeometric distribution test for d . the one-tailed Fisher’s Exact test for ( e ) and ( f ). Two-tailed Student’s t -test for ( m ), ( n ), ( k ), and ( l ). MR, M-CSF + RANKL. All experiments were performed in triplicates unless otherwise stated. Source data are provided in the Source data file.

Article Snippet: For the BRD9 inhibitor/degrader experiments, the cells were treated with either vehicle or varying concentrations of iBRD9 (Tocris Bioscience, 5591) or dBRD9 (R&D Systems, 6606) twenty-four hours after plated, as well as IFN-β1 cytokine (0.0625 ng/ml, R&D Systems, 8234-MB), DEX (10 −11 −10 −9 M), JQ1 (0.25 μM, Abmole Bioscience, M2167) and ZOL (10 μM, Abmole Bioscience, M5032).

Techniques: Control, Inhibition, Binding Assay, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Western Blot, Plasmid Preparation, Knockdown, One-tailed Test, Two Tailed Test

Journal: Immunity

Article Title: A Genome-wide CRISPR Screen Reveals a Role for the Non-canonical Nucleosome-Remodeling BAF Complex in Foxp3 Expression and Regulatory T Cell Function

doi: 10.1016/j.immuni.2020.06.011

Figure Lengend Snippet: The Three SWI/SNF Complex Assemblies Have Distinct Regulatory Roles for Foxp3 Expression in Treg Cells (A) A diagram showing three different variants of SWI/SNF complexes: BAF, ncBAF, and PBAF. BAF-specific subunits (Arid1a and Dpf1–Dpf3) are colored blue, ncBAF-specific subunits (Brd9, Smarcd1, Gltscr1l, and Gltscr1) are colored orange, and PBAF-specific subunits (Pbrm1, Arid2, Brd7, and Phf10) are colored green. Shared components among complexes are colored gray. Also shown is an immunoprecipitation assay of Arid1a, Brd9, Phf10, and Smarca4 in Treg cells. The co-precipitated proteins were probed for shared subunits (Smarca4, Smarcc1, and Smarcb1), BAF-specific Arid1a, ncBAF-specific Brd9, and PBAF-specific Pbrm1. (B) FACS histogram of Foxp3 expression in Treg cells after sgRNA targeting of the indicated SWI/SNF subunits. (C) MFI of Foxp3 after sgRNA targeting of the indicated SWI/SNF subunits. Data represent mean and standard deviation of biological replicates (n = 3–21). (D) Principal-component analysis of RNA-seq data collected from Treg cells transduced with guides against the indicated SWI/SNF subunits. In cases where two independent guides were used to target a gene, the second guide for targeting the gene is indicated as “-2.” (E) MFI of Foxp3 expression in Treg cells after treatment with DMSO or 0.16–10 μM dBRD9 for 4 days. Data represent mean ± SD. Statistical analyses were performed using unpaired two-tailed Student’s t test (non-significant [ns], p ≥ 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). See also Figure S5 .

Article Snippet: For experiments with Brd9 degradation, Treg cells were treated at day 0 with 2.5 μM dBRD9 (Tocris #6606) and cultured for four days for RNA- and ChIP-seq and 0.16-10 μM treated at day 0 and cultured dBRD9 for four days for Foxp3 MFI, cell viability and cell proliferation assays.

Techniques: Expressing, Immunoprecipitation, Standard Deviation, RNA Sequencing Assay, Transduction, Two Tailed Test

Journal: Immunity

Article Title: A Genome-wide CRISPR Screen Reveals a Role for the Non-canonical Nucleosome-Remodeling BAF Complex in Foxp3 Expression and Regulatory T Cell Function

doi: 10.1016/j.immuni.2020.06.011

Figure Lengend Snippet: Brd9 Deletion Reduces Foxp3 Binding at CNS0 and CNS2 Enhancers and a Subset of Foxp3 Target Sites (A) Genome browser tracks of Smarca4, Brd9, and Phf10 ChIP-seq and ATAC-seq signals as well as Foxp3 ChIP-seq in sgNT, sgFoxp3, sgBrd9, and sgPbrm1 Treg cells and Foxp3 in DMSO- and dBRD9-treated Treg cells (2.5 μM dBRD9 for 4 days). The Foxp3 locus is shown with CNS0 and CNS2 enhancers, indicated as gray ovals. (B) Heatmap of Foxp3, Brd9, Smarca4, and Phf10 ChIP-seq and ATAC-seq signals ± 3 kb, centered on Foxp3-bound sites in Treg, ranked according to Foxp3 read density. (C) Venn diagram of the overlap between ChIP-seq peaks in Treg cells for Brd9, Foxp3, and Phf10 (hypergeometric p of Brd9:Foxp3 overlap = e −27665 , hypergeometric p of PHF10:Foxp3 overlap = e −17185 , hypergeometric p of Brd9:PHF10 overlap = e −14217 ). (D) Heatmap of Foxp3 ChIP-seq signals in sgNT, sgFoxp3, sgBrd9, and sgPbrm1 Treg cells ± 3 kb, centered on Foxp3-bound sites in sgNT, ranked according to read density. (E) Venn diagram of the overlap (hypergeometric p = e −11,653 ) between sites that significantly lose Foxp3 binding (FC 1.5, Poisson p < 0.0001) in sgFoxp3 and sgBrd9, overlaid on all Foxp3-bound sites in sgNT (gray). (F) Histogram of Foxp3 ChIP read density ± 1 kb surrounding the peak center of sites that significantly lose Foxp3 binding in sgFoxp3 and sgBrd9 (n = 1,699) in sgNT, sgFoxp3, sgBrd9, and sgPbrm1. (G) As in (E) but for sites that lose H3K27ac (FC 1.5, Poisson p < 0.0001, hypergeometric p of overlap = e −7,938 ). (H) As in (F) but for H3K27ac ChIP read density. (I) As in (D) but for Foxp3 ChIP-seq signals in DMSO- and dBRD9-treated Treg cells at all Foxp3-bound sites in DMSO. (J) As in (E), but for sites that significantly lose Foxp3 binding in dBRD9 treated Treg cells versus DMSO (FC 1.5, Poisson p < 0.0001). (K) As in (F) but for DMSO- and dBRD9-treated cells. (L) As in (F) but for Treg cells transduced with sgNT or sgBrd9, with ectopic expression of the MIGR vector control or Foxp3. See also Figure S6 .

Article Snippet: For experiments with Brd9 degradation, Treg cells were treated at day 0 with 2.5 μM dBRD9 (Tocris #6606) and cultured for four days for RNA- and ChIP-seq and 0.16-10 μM treated at day 0 and cultured dBRD9 for four days for Foxp3 MFI, cell viability and cell proliferation assays.

Techniques: Binding Assay, ChIP-sequencing, Transduction, Expressing, Plasmid Preparation, Control

Journal: Immunity

Article Title: A Genome-wide CRISPR Screen Reveals a Role for the Non-canonical Nucleosome-Remodeling BAF Complex in Foxp3 Expression and Regulatory T Cell Function

doi: 10.1016/j.immuni.2020.06.011

Figure Lengend Snippet: Brd9 Co-regulates the Expression of Foxp3 and a Subset of Foxp3 Target Genes (A) Volcano plot of log2 fold change RNA expression in sgFoxp3 versus sgNT Treg cells versus adjusted p value (Benjamini-Hochberg). The numbers of down and up genes are indicated and colored blue and red, respectively. (B) Significance of enrichment of Foxp3-dependent genes in each Gene Ontology. (C) Pie chart of Foxp3 and Brd9 binding by ChIP-seq for Foxp3-dependent genes. (D) Gene set enrichment analysis (GSEA) enrichment plot for up and down genes in sgBrd9 versus sgNT compared with RNA-seq data of genes that significantly change in sgFoxp3 versus sgNT Treg cells. ES, enrichment score; NES, normalized enrichment score; FWER, family-wise error rate. (E) As in (D) but for up and down genes in dBRD9 versus DMSO Treg cells. (F) GSEA of the sgFoxp3 versus sgNT RNA-seq data; the plot shows the FWER p value versus the NES. See also Figure S6 and .

Article Snippet: For experiments with Brd9 degradation, Treg cells were treated at day 0 with 2.5 μM dBRD9 (Tocris #6606) and cultured for four days for RNA- and ChIP-seq and 0.16-10 μM treated at day 0 and cultured dBRD9 for four days for Foxp3 MFI, cell viability and cell proliferation assays.

Techniques: Expressing, RNA Expression, Binding Assay, ChIP-sequencing, RNA Sequencing Assay

Journal: Immunity

Article Title: A Genome-wide CRISPR Screen Reveals a Role for the Non-canonical Nucleosome-Remodeling BAF Complex in Foxp3 Expression and Regulatory T Cell Function

doi: 10.1016/j.immuni.2020.06.011

Figure Lengend Snippet: The ncBAF Complex Regulates Treg Cell Suppressor Function In Vitro and In Vivo (A) In vitro suppression assay of Treg cells with sgRNA targeting of Brd9 , Smarcd1 , Pbrm1 , and Phf10 . sgNT was used as a non-targeting control (n = 3 per group; data represent ± SD). (B) In vitro suppression assay of sgBrd9 or sgNT with ectopic expression of Foxp3 or the control vector MIGR (n = 3 per group; data represent ± SD). (C–F) Experiment to measure function of sgNT or sgBrd9 Treg cells relative to “no Treg” cells in a T cell transfer-induced colitis model. (C) Experimental procedure. (D) Body weight loss. (E) Colon histology (left) and colitis scores (right). (F) Percentage of Foxp3 + cells in the transferred CD45.2 + CD4 + Treg population at the endpoint (n = 4–7 per group; data represent mean ± SEM). Statistical analyses were performed using unpaired two-tailed Student’s t test (ns, p ≥ 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). See also Figure S7 .

Article Snippet: For experiments with Brd9 degradation, Treg cells were treated at day 0 with 2.5 μM dBRD9 (Tocris #6606) and cultured for four days for RNA- and ChIP-seq and 0.16-10 μM treated at day 0 and cultured dBRD9 for four days for Foxp3 MFI, cell viability and cell proliferation assays.

Techniques: In Vitro, In Vivo, Suppression Assay, Control, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Immunity

Article Title: A Genome-wide CRISPR Screen Reveals a Role for the Non-canonical Nucleosome-Remodeling BAF Complex in Foxp3 Expression and Regulatory T Cell Function

doi: 10.1016/j.immuni.2020.06.011

Figure Lengend Snippet: Targeting Brd9 in Treg Cells Improves Anti-tumor Immunity (A) Experimental procedure to measure the function of sgNT or sgBrd9 Treg cells relative to “no Treg” cells in the MC38 tumor model. (B) Tumor growth curve. (C) Tumor weight at the endpoint. (D and E) Bar graph of total CD4 T cell (D) and CD8 T cell (E) percentage in the CD45 + immune cell population. (F and G) Bar graph of the IFN-γ + cell percentage in CD4 T cells (F) and CD8 T cells (G). (H) Bar graph of the CD4 + GFP + Foxp3 + donor cell percentage in CD4 T cells. (I) Ratio of CD8:Treg cells. (J) Bar graph of the Foxp3 – ex-Treg cell percentage in the transferred Treg population marked by the GFP reporter. (K) Bar graph of Foxp3 – IFN-γ + cell percentage in the transferred Treg population (n = 5–7 per group; data represent mean ± SEM). Statistical analyses were performed using unpaired two-tailed Student’s t test (ns, p ≥ 0.05; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). See also Figure S7 .

Article Snippet: For experiments with Brd9 degradation, Treg cells were treated at day 0 with 2.5 μM dBRD9 (Tocris #6606) and cultured for four days for RNA- and ChIP-seq and 0.16-10 μM treated at day 0 and cultured dBRD9 for four days for Foxp3 MFI, cell viability and cell proliferation assays.

Techniques: Two Tailed Test

Journal: Immunity

Article Title: A Genome-wide CRISPR Screen Reveals a Role for the Non-canonical Nucleosome-Remodeling BAF Complex in Foxp3 Expression and Regulatory T Cell Function

doi: 10.1016/j.immuni.2020.06.011

Figure Lengend Snippet:

Article Snippet: For experiments with Brd9 degradation, Treg cells were treated at day 0 with 2.5 μM dBRD9 (Tocris #6606) and cultured for four days for RNA- and ChIP-seq and 0.16-10 μM treated at day 0 and cultured dBRD9 for four days for Foxp3 MFI, cell viability and cell proliferation assays.

Techniques: Recombinant, Transfection, Software

Journal: Nature Communications

Article Title: BAF60a deficiency uncouples chromatin accessibility and cold sensitivity from white fat browning

doi: 10.1038/s41467-020-16148-1

Figure Lengend Snippet: a qPCR analysis of gene expression during brown adipocyte differentiation following transduction of vector or Cre retrovirus ( n = 3 per group). b Immunoblots of brown adipocyte lysates during differentiation as in a . c qPCR analysis of differentiated brown adipocyte gene expression following 5 h of treatment with vehicle (Veh, n = 3, filled) or norepinephrine (NE, n = 3, open). d MitoTracker staining (scale bar = 100 μm; n = 3 per group). e Oxygen consumption rate (OCR) before and after FCCP addition ( n = 6 per group). Data in a , c , e represent mean ± s.d. ** P < 0.01, Vec vs. Cre; two-tailed unpaired Student’s t test. f Immunoblots of BAF60a-associated proteins in brown adipocytes (representative of three experiments). g Immunoblots of brown adipocyte lysates during differentiation in the presence of DMSO or 250 nM dBRD9 ( n = 2 per group). h qPCR analysis of Ucp1 expression ( n = 3 per group). i Immunoblots of brown adipocyte lysates. Differentiated adipocytes were treated with Veh or NE in the presence of DMSO, I-BRD9 (1 or 10 μM), or dBRD9 (100 or 500 nM) ( n = 2 per group). j qPCR analysis of Ucp1 expression in brown adipocytes treated with Veh or NE in the presence of DMSO, 10 μM I-BRD9 or 500 nM dBRD9 ( n = 4 per group). Data in h , j represent mean ± s.d. * P < 0.05, ** P < 0.01, DMSO vs. I-BRD9 or dBRD9; two-tailed unpaired Student’s t test. Source data are provided as a Source Data file.

Article Snippet: For NE stimulation, differentiated adipocytes were incubated with DMSO, I-BRD9 (Cayman), or dBRD9 for 6 h before the addition of saline or 1 μM NE for another 4 h (RNA) or 6 h (protein).

Techniques: Gene Expression, Transduction, Plasmid Preparation, Western Blot, Staining, Two Tailed Test, Expressing

Journal: Nature Communications

Article Title: BAF60a deficiency uncouples chromatin accessibility and cold sensitivity from white fat browning

doi: 10.1038/s41467-020-16148-1

Figure Lengend Snippet: a qPCR analysis of iWAT gene expression in mice following cold acclimation (flox, n = 6, filled; AKO, n = 9, open). Data represent mean ± s.e.m.; two-tailed unpaired Student’s t test, flox vs. AKO. b qPCR analysis of gene expression in differentiated beige adipocytes after treatments (5 h) with Veh, ACTH, KISS1, or Oxytocin (OXT) at two doses ( n = 4 per group). Two-tailed unpaired Student’s t test, treatments vs. Veh. c qPCR analysis of Mc2r expression in stromal vascular fraction (SVF) and mature adipocytes (Adipocyte) from iWAT of flox ( n = 4, filled) and AKO ( n = 3, open) mice. Data represent mean ± s.e.m. flox vs. AKO, two-tailed unpaired Student’s t test. d UCSC genome browser view of ATAC-seq peaks at the Mc2r locus. e qPCR analysis of Mc2r gene expression in beige adipocytes after 5 h treatments with DMSO or 1 μM rosiglitazone (Rosi) ( n = 4 per group). Data represent mean ± s.d. Rosi vs. DMSO, two-tailed unpaired Student’s t test. f Norepinephrine (NE) and epinephrine (Epi) levels in iWAT after chronic cold acclimation (flox, n = 10, filled; AKO, n = 10, open). Plasma ACTH levels at ambient temperature (flox n = 7; AKO, n = 6) or following cold acclimation (flox n = 6; AKO n = 7). Data represent mean ± s.e.m. Source data are provided as a Source Data file.

Article Snippet: For NE stimulation, differentiated adipocytes were incubated with DMSO, I-BRD9 (Cayman), or dBRD9 for 6 h before the addition of saline or 1 μM NE for another 4 h (RNA) or 6 h (protein).

Techniques: Gene Expression, Two Tailed Test, Expressing, Clinical Proteomics