Journal: Cancer Science

Article Title: Tumor suppressive role of the epigenetic master regulator BRD3 in colorectal cancer

doi: 10.1111/cas.16129

Figure Lengend Snippet: Effects of BRD3 knockdown and overexpression on cell proliferation in colorectal cancer (CRC) cells in vitro and in vivo. (A) BRD3 mRNA expression normalized to 18S expression according to reverse‐transcription quantitative PCR (RT‐qPCR) and protein expression according to western blot analysis (WB) in BRD3 ‐knockdown and control CRC cells. ** p < 0.01; *** p < 0.001. (B) Colony formation assays using BRD3 ‐knockdown CRC cells. ** p < 0.01. (C) BRD3 mRNA expression normalized to 18S expression according to RT‐qPCR and protein expression according to WB in BRD3 transiently overexpressing and control CRC cells. ** p < 0.01. (D) Colony formation assays using BRD3 transiently overexpressing CRC cells. * p < 0.05; ** p < 0.01. (E) BRD3 mRNA expression normalized to 18S expression according to RT‐qPCR (left) and protein expression according to WB (right) in CRC cells with stable BRD3 overexpression and control CRC cells. ** p < 0.01. (F) In vivo analysis using a murine xenograft model. Size of tumors derived from CRC cells with stable BRD3 overexpression and control CRC cells. N = 9 per group. * p < 0.05; ** p < 0.01; *** p < 0.001. (G) BRD3 mRNA expression normalized to 18S expression according to RT‐qPCR (left) and immunohistochemical staining of BRD3 (right) in tumors derived from CRC cells with stable BRD3 overexpression and control CRC cells. ** p < 0.01. Scale bars, 20 μm; original magnification, ×200.

Article Snippet: The following antigen‐specific primary antibodies were used: rabbit polyclonal antibody against BRD3 (1:1000, Proteintech), rabbit monoclonal antibody against BRD4 (1: 2000, Abcam), the mixture of three specific rabbit monoclonal antibodies against phosphor‐cdk2 Tyr15, phosphor‐histone H3 Ser10, and β‐actin (1:250, ab136810, Abcam), rabbit polyclonal antibody against Lamin B1 (1:5000, 12987‐1‐AP, Proteintech), and mouse monoclonal antibody against β‐actin (1:1000, Santa Cruz Biotechnology).

Techniques: Knockdown, Over Expression, In Vitro, In Vivo, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Control, Derivative Assay, Immunohistochemical staining, Staining

Journal: Cancer Science

Article Title: Tumor suppressive role of the epigenetic master regulator BRD3 in colorectal cancer

doi: 10.1111/cas.16129

Figure Lengend Snippet: Cell cycle progression halted by BRD3 from the G1 to the S phase in colorectal cancer (CRC) cells. (A) Protein expression using western blot analysis (WB) for BRD3 and Cdk2 pTyr15 in BRD3‐ knockdown (left) and transiently overexpressing (right) CRC cells. (B) BRD3 mRNA expression normalized to 18S expression according to RT‐qPCR (left), and protein expression according to WB (right) in CRC cells with BRD3 stable overexpression and control CRC cells. *** p < 0.001. (C) Cell cycle analysis by flow cytometry after stimulation with FBS in CRC cells with BRD3 stable overexpression and control CRC cells. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The following antigen‐specific primary antibodies were used: rabbit polyclonal antibody against BRD3 (1:1000, Proteintech), rabbit monoclonal antibody against BRD4 (1: 2000, Abcam), the mixture of three specific rabbit monoclonal antibodies against phosphor‐cdk2 Tyr15, phosphor‐histone H3 Ser10, and β‐actin (1:250, ab136810, Abcam), rabbit polyclonal antibody against Lamin B1 (1:5000, 12987‐1‐AP, Proteintech), and mouse monoclonal antibody against β‐actin (1:1000, Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Knockdown, Quantitative RT-PCR, Over Expression, Control, Cell Cycle Assay, Flow Cytometry

Journal: Cancer Science

Article Title: Tumor suppressive role of the epigenetic master regulator BRD3 in colorectal cancer

doi: 10.1111/cas.16129

Figure Lengend Snippet: Upregulated p21 expression by BRD3 in colorectal cancer (CRC) cells. (A) p21 mRNA expression normalized to 18S expression according to reverse‐transcription quantitative PCR (RT‐qPCR) in BRD3 ‐knockdown and control CRC cells. ** p < 0.01; *** p < 0.001. (B) p21 mRNA expression normalized to 18S expression according to RT‐qPCR in BRD3 transiently overexpressing and control CRC cells. * p < 0.05; *** p < 0.001. (C) Protein expression using western blot analysis (WB) for BRD3, p21, LaminB1 in BRD3 ‐knockdown (left) and transiently overexpressing (right) CRC cells. (D) p21 and BRD4 mRNA expression normalized to 18S expression according to RT‐qPCR in tumor tissues derived from CRC cells with BRD3 stable overexpression and in those derived from control CRC cells. *** p < 0.001. (E) Immunohistochemical staining of BRD4 or p21 in tumor tissues derived from CRC cells with BRD3 stable overexpression and in those derived from control CRC cells. Scale bars, 20 μm; original magnification, ×200. p21 scores were determined by observing the most intensely stained areas. ** p < 0.01.

Article Snippet: The following antigen‐specific primary antibodies were used: rabbit polyclonal antibody against BRD3 (1:1000, Proteintech), rabbit monoclonal antibody against BRD4 (1: 2000, Abcam), the mixture of three specific rabbit monoclonal antibodies against phosphor‐cdk2 Tyr15, phosphor‐histone H3 Ser10, and β‐actin (1:250, ab136810, Abcam), rabbit polyclonal antibody against Lamin B1 (1:5000, 12987‐1‐AP, Proteintech), and mouse monoclonal antibody against β‐actin (1:1000, Santa Cruz Biotechnology).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Knockdown, Control, Western Blot, Derivative Assay, Over Expression, Immunohistochemical staining, Staining

Journal: Cancer Science

Article Title: Tumor suppressive role of the epigenetic master regulator BRD3 in colorectal cancer

doi: 10.1111/cas.16129

Figure Lengend Snippet: Expression of BET family genes and correlations between BRD3 and other BET gene expression levels in colorectal cancer (CRC). (A) mRNA expression of BET family genes ( BRD3 , BRD2 , BRD4 , BRDT ) in 380 CRC tissues and 51 normal colon tissues obtained from The Cancer Genome Atlas (TCGA) dataset. *** p < 0.001. (B) BRD3 and BRD4 mRNA expression according to reverse‐transcription quantitative PCR in 144 CRC tissues and paired normal colon tissues in our CRC cohort dataset. *** p < 0.001. (C, D) Correlation between the mRNA expression of BRD3 and that of other BET genes in TCGA dataset (A) and our CRC cohort (B). R indicates the Pearson correlation coefficient. (E) Immunohistochemical staining of BRD3 and BRD4. Upper: scale bar, 200 μm (left) and 20 μm (right). Original magnification, ×40 (left) and ×400 (right). Lower: scale bar, 200 μm (upper) and 20 μm (lower). N, normal tissue; T, tumor tissue.

Article Snippet: The following antigen‐specific primary antibodies were used: rabbit polyclonal antibody against BRD3 (1:1000, Proteintech), rabbit monoclonal antibody against BRD4 (1: 2000, Abcam), the mixture of three specific rabbit monoclonal antibodies against phosphor‐cdk2 Tyr15, phosphor‐histone H3 Ser10, and β‐actin (1:250, ab136810, Abcam), rabbit polyclonal antibody against Lamin B1 (1:5000, 12987‐1‐AP, Proteintech), and mouse monoclonal antibody against β‐actin (1:1000, Santa Cruz Biotechnology).

Techniques: Expressing, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining

Journal: Cancer Science

Article Title: Tumor suppressive role of the epigenetic master regulator BRD3 in colorectal cancer

doi: 10.1111/cas.16129

Figure Lengend Snippet: BRD3 expression and enrichment pathway analysis in single‐cell colorectal cancer (CRC) data. (A) Uniform Manifold Approximation and Projection (UMAP) of cell types in all cells (left) and tissues (right). (B) UMAP of cell types after annotation of carcinoma cells. (C) Dot plot of the expression and expression proportions of BRD3 , BRD4 , and BRD2 per cell type. The circle size represents the cell proportion. (D) Expression of BRD3 in UMAP representations. (E) Upper: UMAP distribution in carcinoma epithelial cells with high versus low BRD3 expression. Lower: violin plots of BRD3 expression in high versus low BRD3 mRNA expression groups (divided by the median BRD3 mRNA level). (F) Reactome pathway analysis of carcinoma epithelial cells with high versus low BRD3 expression.

Article Snippet: The following antigen‐specific primary antibodies were used: rabbit polyclonal antibody against BRD3 (1:1000, Proteintech), rabbit monoclonal antibody against BRD4 (1: 2000, Abcam), the mixture of three specific rabbit monoclonal antibodies against phosphor‐cdk2 Tyr15, phosphor‐histone H3 Ser10, and β‐actin (1:250, ab136810, Abcam), rabbit polyclonal antibody against Lamin B1 (1:5000, 12987‐1‐AP, Proteintech), and mouse monoclonal antibody against β‐actin (1:1000, Santa Cruz Biotechnology).

Techniques: Expressing

Journal: Cancer Science

Article Title: Tumor suppressive role of the epigenetic master regulator BRD3 in colorectal cancer

doi: 10.1111/cas.16129

Figure Lengend Snippet: Spatial transcriptomic and single‐cell analysis revealing a positive correlation between BRD3 and p21 expression in colorectal cancer (CRC). (A) Pathological diagnosis in colorectal tissue in a tissue slide used for spatial transcriptomic analysis. Normal: normal tissue; Tumor: tumor tissue. (B) Spatial distribution of BRD3 , BRD4 , and p21 expression. (C) Upper: spatial distribution in tissue regions with high versus low BRD3 expression. Lower: violin plots of BRD3 expression in high versus low BRD3 mRNA expression groups (divided by the median BRD3 mRNA level). *** p < 0.001. (D) Violin plots of BRD4 and p21 expression in the high versus low BRD3 mRNA expression groups. * p < 0.05. (E) Gene set enrichment analysis of tissue regions with high versus low BRD3 expression. Pval, p ‐value; FDR, false discovery rate; NES, normalized enrichment score. (F) p21 expression in Uniform Manifold Approximation and Projection (UMAP) representations of carcinoma epithelial cells based on scRNA‐seq data (left), and violin plots of p21 expression in the high versus low BRD3 mRNA expression groups (right). *** p < 0.001.

Article Snippet: The following antigen‐specific primary antibodies were used: rabbit polyclonal antibody against BRD3 (1:1000, Proteintech), rabbit monoclonal antibody against BRD4 (1: 2000, Abcam), the mixture of three specific rabbit monoclonal antibodies against phosphor‐cdk2 Tyr15, phosphor‐histone H3 Ser10, and β‐actin (1:250, ab136810, Abcam), rabbit polyclonal antibody against Lamin B1 (1:5000, 12987‐1‐AP, Proteintech), and mouse monoclonal antibody against β‐actin (1:1000, Santa Cruz Biotechnology).

Techniques: Single-cell Analysis, Expressing, Biomarker Discovery

Journal: Cancer Science

Article Title: Tumor suppressive role of the epigenetic master regulator BRD3 in colorectal cancer

doi: 10.1111/cas.16129

Figure Lengend Snippet: Tumor growth inhibited by BRD3 overexpression in BRD4 ‐knockdown colorectal cancer (CRC) cells. (A) BRD4 mRNA expression normalized to 18S expression according to reverse‐transcription quantitative PCR (RT‐qPCR) (upper) and protein expression according to western blot analysis (WB) (lower) in BRD4 ‐knockdown and control CRC cells. * p < 0.05; ** p < 0.01. (B) Colony formation assays using BRD4 ‐knockdown cells. * p < 0.05. ** p < 0.01. (C) Colony formation assays after BRD4 knockdown in CRC cells with BRD3 stable overexpression and control CRC cells. * p < 0.05; ** p < 0.01; *** p < 0.001. (D) Summary of the results. BRD3 inhibits proliferation of CRC cells by suppressing cell cycle progression possibly via promoting p21 expression.

Article Snippet: The following antigen‐specific primary antibodies were used: rabbit polyclonal antibody against BRD3 (1:1000, Proteintech), rabbit monoclonal antibody against BRD4 (1: 2000, Abcam), the mixture of three specific rabbit monoclonal antibodies against phosphor‐cdk2 Tyr15, phosphor‐histone H3 Ser10, and β‐actin (1:250, ab136810, Abcam), rabbit polyclonal antibody against Lamin B1 (1:5000, 12987‐1‐AP, Proteintech), and mouse monoclonal antibody against β‐actin (1:1000, Santa Cruz Biotechnology).

Techniques: Over Expression, Knockdown, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Control

Journal: Cell reports

Article Title: Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins

doi: 10.1016/j.celrep.2020.108532

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-BRD3 , Bethyl Laboratories , Cat# A302–368A; RRID: AB_1907251.

Techniques: Recombinant, Staining, Cell Viability Assay, Cell Culture, Gel Extraction, Gene Expression, Control, CRISPR, Knock-Out, Activation Assay, Software