cdna of full-length brd4  (Addgene inc)

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: p113 physically interacts with ZRF1 and BRD4 in NB cells. a Volcano plots showing differentially expressed genes (fold change> 1.5, P < 0.05) in SH-SY5Y cells stably transfected with empty vector (mock) or ecircCUX1 . b Coomassie blue staining (left panel) and Venn diagram (right panel) revealing identification of p113-interacting proteins pulled down by p113 or Flag-tag antibody in SH-SY5Y cells stably transfected with 3Flag-tagged p113 , and those overlapped with transcription factors (TF) or epigenetic factors derived from ChIP-X and EpiFactors databases. c Co-IP and western blot assays indicating the interaction among p113, ZRF1, and BRD4 in SH-SY5Y and BE(2)-C cells stably transfected with mock, ecircCUX1 , scramble shRNA (sh-Scb), or sh-ecircCUX1. d Secondary co-IP assays showing protein interaction among p113, ZRF1, and BRD4 in SH-SY5Y cells stably transfected with HA-tagged p113 , Flag-tagged ZRF1 , and His-tagged BRD4 . e BiFC assay revealing the interaction of p113 with ZRF1 or BRD4 (arrowheads) in SH-SY5Y cells stably transfected with indicated constructs, with nuclei stained by DAPI. Scale bars: 10 μm. f and g Western blot assay (g) validating the knockdown of ZRF1 or BRD4 in SH-SY5Y cells stably transfected with scramble (Scb) or specific sgRNA for CRISPR interference (CRISPRi, f). Wild type (WT) cells were taken as negative controls. h Co-IP and western blot assays indicating the interaction of p113 with ZRF1 or BRD4 in SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with CRISPRi sgRNA specific against ZRF1 or BRD4 . i Schematic illustration of protein interaction among p113, ZRF1, and BRD4. j Dual-luciferase assay showing the activity of ZRF1 in NB cells stably transfected with mock, ecircCUX1 , ecircCUX1 Mut, p113 , sh-Scb, or sh-ecircCUX1 ( n = 5). Fisher’s exact test for overlapping analysis in b . ANOVA compared the difference in j . * P < 0.05 vs. mock or sh-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in c - e , g , h and j

Article Snippet: Human p113 ORF (342 bp), ZRF1 cDNA (1866 bp), and BRD4 cDNA (4089 bp) were subcloned into pBiFC-VN173 or pBiFC-VC155 (Addgene), and co-transfected into tumor cells with Lipofectamine 2000 (Invitrogen) for 24 h. The fluorescence was observed with a confocal microscope (Nikon, Japan) [ , , ].

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Staining, FLAG-tag, Derivative Assay, Co-Immunoprecipitation Assay, Western Blot, shRNA, Bimolecular Fluorescence Complementation Assay, Construct, CRISPR, Luciferase, Activity Assay

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: p113/ZRF1/BRD4 complex promotes lipid metabolic reprogramming and mitochondrial complex I activity in NB cells. a Heatmap, distribution, and binding motif of ChIP-Seq (left panel) assay revealing genomic enrichment of ZRF1 in SH-SY5Y cells, while Venn diagram, heatmap, and GO pathway (right panel) showing identification of p113/ZRF1/BRD4 target genes by overlapping analysis of RNA-seq results upon p113 over-expression and ChIP-seq peaks of ZRF1 or BRD4. b ChIP-seq assay showing the binding peak of BRD4 or ZRF1 on promoter regions of ALDH3A1 , NDUFA1 , or NDUFAF5 in SH-SY5Y cells. c Western blot assay indicating the expression of ALDH3A1, NDUFA1, or NDUFAF5 in SH-SY5Y cells stably transfected with empty vector (mock) or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 or BRD4 for CRISPRi. d Schematic illustration showing the involvement of ALDH3A1, NDUFA1, or NDUFAF5 in lipid metabolic reprogramming and mitochondrial respiratory activity. e Relative OCR levels in SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 or BRD4 for CRISPRi ( n = 5). f Relative fatty acid levels, complex I activity, NAD + /NADH ratio, and ATP levels in SH-SY5Y cells stably transfected with mock or ecircCUX1 , and those co-transfected with sgRNA specific against ZRF1 or BRD4 for CRISPRi ( n = 5). ANOVA compared the difference in e and f . * P < 0.05 vs. mock+CRISPRi-Scb. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in c , e and f

Article Snippet: Human p113 ORF (342 bp), ZRF1 cDNA (1866 bp), and BRD4 cDNA (4089 bp) were subcloned into pBiFC-VN173 or pBiFC-VC155 (Addgene), and co-transfected into tumor cells with Lipofectamine 2000 (Invitrogen) for 24 h. The fluorescence was observed with a confocal microscope (Nikon, Japan) [ , , ].

Techniques: Activity Assay, Binding Assay, ChIP-sequencing, RNA Sequencing Assay, Over Expression, Western Blot, Expressing, Stable Transfection, Transfection, Plasmid Preparation

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: Therapeutic blocking p113-ZRF1 interaction inhibits NB progression. a 3D structure and sequences of inhibitory peptides (ZIP-12) for blocking interaction between p113 and ZRF1, and those of mutant control (Ctrl) peptides. b Confocal images showing the distribution of synthesized FITC-labeled Ctrl or ZIP-12 peptides (20 μmol·L − 1 , arrowheads) within cultured BE(2)-C cells, with nuclei and cellular membranes staining with DAPI or Dil. Scale bars: 10 μm. c Co-IP and western blot assays indicating the interaction of p113 with ZRF1 or BRD4 in BE(2)-C cells treated with Ctrl or ZIP-12 peptides (20 μmol·L − 1 ) for 24 h. d Relative fatty acid levels, complex I activity, NAD + /NADH ratio, and ATP levels in BE(2)-C cells treated with Ctrl or ZIP-12 peptides (20 μmol·L − 1 ) for 24 h. e In vivo images (left upper panel), growth curve (right panel), and weight at the end points (right panel) of xenografts formed by subcutaneous injection of BE(2)-C cells in nude mice ( n = 5 per group) that were treated with intravenous injection of Ctrl or ZIP-12 peptides (5 mg·kg − 1 ) as indicated (left lower panel). f In vivo imaging (left panel), lung metastatic colonization (right lower panel), and Kaplan–Meier curves (right lower panel) of nude mice ( n = 5 for each group) treated with tail vein injection of BE(2)-C cells, Ctrl or ZIP-12 peptides (5 mg·kg − 1 ) as indicated (right upper panel). Student’s t test or ANOVA compared the difference in d - f . Log-rank test for survival comparison in f . * P < 0.05, ** P < 0.01 vs. Ctrl. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in b - d

Article Snippet: Human p113 ORF (342 bp), ZRF1 cDNA (1866 bp), and BRD4 cDNA (4089 bp) were subcloned into pBiFC-VN173 or pBiFC-VC155 (Addgene), and co-transfected into tumor cells with Lipofectamine 2000 (Invitrogen) for 24 h. The fluorescence was observed with a confocal microscope (Nikon, Japan) [ , , ].

Techniques: Blocking Assay, Mutagenesis, Synthesized, Labeling, Cell Culture, Staining, Co-Immunoprecipitation Assay, Western Blot, Activity Assay, In Vivo, Injection, In Vivo Imaging

Journal: Molecular Cancer

Article Title: p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation

doi: 10.1186/s12943-021-01421-8

Figure Lengend Snippet: CUX1 , ZRF1 , BRD4 and target genes are associated with poor outcome of NB patients. a Kaplan–Meier curves indicating overall survival of 498 well-defined NB cases (GSE62564) with high or low expression of CUX1 (cutoff value = 32.233), ZRF1 (cutoff value = 21.749), BRD4 (cutoff value = 652.58), ALDH3A1 (cutoff value = 1.181), NDUFA1 (cutoff value = 22.511), or NDUFAF5 (cutoff value = 7.964). b The positive expression correlation between ZRF1 and ALDH3A1 , NDUFA1 , or NDUFAF5 in 498 well-defined NB cases (GSE62564). c The mechanisms underlying p113-faciliated NB progression: as a novel protein encoded by ecircCUX1 , p113 cooperates with ZRF1 and BRD4 to activate the transcription of ALDH3A1 , NDUFA1 , or NDUFAF5 , resulting in promoted conversion of fatty aldehydes into fatty acids, fatty acid β-oxidation, mitochondrial complex I activity, growth, and aggressiveness of NB cells. Meanwhile, inhibitory peptides (ZIP-12) blocking p113-ZRF1 interaction suppresses tumor progression. Log-rank test for survival comparison in a . Pearson’s correlation coefficient for b

Article Snippet: Human p113 ORF (342 bp), ZRF1 cDNA (1866 bp), and BRD4 cDNA (4089 bp) were subcloned into pBiFC-VN173 or pBiFC-VC155 (Addgene), and co-transfected into tumor cells with Lipofectamine 2000 (Invitrogen) for 24 h. The fluorescence was observed with a confocal microscope (Nikon, Japan) [ , , ].

Techniques: Expressing, Activity Assay, Blocking Assay

Journal: Scientific Reports

Article Title: BRD4 regulates adiponectin gene induction by recruiting the P-TEFb complex to the transcribed region of the gene

doi: 10.1038/s41598-017-12342-2

Figure Lengend Snippet: Effects of BRD4 depletion and inhibition on mRNA and protein expression of Adipoq and lipid droplet accumulation in 3T3-L1 adipocytes. ( a ) mRNA levels of Brd4 and protein levels of BRD4, BRD2, TRAP220, CYCLIN T1, CDK9, PPARγ2, and TATA box-binding protein (TBP) at 2 and 8 days after differentiation stimulation. Control or Brd4 shRNA-expressing 3T3-L1 cells were treated with medium for differentiation. ( b ) Levels of Adipoq mRNA in cells and ADIPOQ protein in medium at 2 and 8 days after differentiation stimulation. ( c ) Lipid droplet accumulation at 5 days after differentiation stimulation (Oil red analysis). ( d ) Adipoq mRNA in 3T3-L1 adipocytes, secreted ADIPOQ protein in the medium, and protein levels of BRD4, BRD2 and TBP in cells. (+)-JQ1 treatment of 3T3-L1 adipocytes was performed for 4 days from the adipocyte differentiation. ( e ) Lipid droplet accumulation (Oil red O analysis). The data shown are means ± SEM of 6 wells per condition. ** P < 0.01 versus the corresponding control cells by Student’s t -test ( a , b ) or Dunnett’s test based on analysis of variance ( d ).

Article Snippet: For preparation of BRD4-overexpressing cells, a mouse Brd4 cDNA (Unigene ID Mm.

Techniques: Inhibition, Expressing, Binding Assay, Control, shRNA

Journal: Scientific Reports

Article Title: BRD4 regulates adiponectin gene induction by recruiting the P-TEFb complex to the transcribed region of the gene

doi: 10.1038/s41598-017-12342-2

Figure Lengend Snippet: Effects of BRD4 depletion on binding of BRD4, acetylated histones, P-TEFb, PPARγ2, and TRAP220 around the Adipoq gene in 3T3-L1 adipocytes. Control or Brd4 shRNA-expressing 3T3-L1 cells were treated with medium for differentiation. ChIP assays for BRD4, acetylated histone H3, acetylated histone H4, CDK9, PPARγ2, TRAP220, and normal IgG were performed at 2 and 8 days after differentiation. The data shown are means ± SEM of 6 wells per condition in a single experiment. * P < 0.05, ** P < 0.01 versus the corresponding control cells by Student’s t -test.

Article Snippet: For preparation of BRD4-overexpressing cells, a mouse Brd4 cDNA (Unigene ID Mm.

Techniques: Binding Assay, Control, shRNA, Expressing

Journal: Scientific Reports

Article Title: BRD4 regulates adiponectin gene induction by recruiting the P-TEFb complex to the transcribed region of the gene

doi: 10.1038/s41598-017-12342-2

Figure Lengend Snippet: Effects of BRD4 depletion on expression of genes related to insulin sensitivity in 3T3-L1 adipocytes. Control or Brd4 shRNA-expressing 3T3-L1 cells were treated with medium for differentiation and real-time RT-PCR analyses for Albp , Glut4 , Fas , Accα , Accβ , Dgat1 , Lpl , Hsl , Aco , Pparγ1 , Pparγ2 , Creb , C/ebpα , C/ebpβ , C/ebpδ , C/ebpγ , C/ebpζ , Chrebp , Pgc1α , Srebp1 , Srebp1α , Lxrα and Lxrβ were performed at 2 and 8 days after differentiation stimulation. The data shown are means ± SEM of 6 wells per condition in a single experiment. * P < 0.05, ** P < 0.01 versus the corresponding control cells by Student’s t -test.

Article Snippet: For preparation of BRD4-overexpressing cells, a mouse Brd4 cDNA (Unigene ID Mm.

Techniques: Expressing, Control, shRNA, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: BRD4 regulates adiponectin gene induction by recruiting the P-TEFb complex to the transcribed region of the gene

doi: 10.1038/s41598-017-12342-2

Figure Lengend Snippet: Effects of BRD4 overexpression on Brd4 mRNA and protein levels and mRNA levels of genes related to insulin sensitivity in 3T3-L1 adipocytes. Brd4 -overexpressing or control plasmid transfected 3T3-L1 cells were treated with medium for differentiation. Immunoblot analysis for BRD4, CYCLIN T1, CDK9, PPARγ2 and TBP and real-time RT-PCR analyses for Brd4 , Adipoq , Albp , Glut4 , Fas , Accα , Accβ , Dgat1 , Lpl , Hsl , Aco , Pparγ1 , Pparγ2 , Creb , C/ebpα , C/ebpβ , C/ebpδ , C/ebpγ , C/ebpζ , Chrebp , Pgc1α , Srebp1 , Srebp1α , Lxrα and Lxrβ were performed in samples at 2 days after differentiation. The data shown are means ± SEM of 6 wells per condition in a single experiment. * P < 0.05, ** P < 0.01 versus the corresponding control cells by Student’s t -test.

Article Snippet: For preparation of BRD4-overexpressing cells, a mouse Brd4 cDNA (Unigene ID Mm.

Techniques: Over Expression, Control, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: BRD4 regulates adiponectin gene induction by recruiting the P-TEFb complex to the transcribed region of the gene

doi: 10.1038/s41598-017-12342-2

Figure Lengend Snippet: Effects of Brd4 genetic depletion on Adipoq and fatty acid synthesis-related gene expression in mesenteric fat tissues and ADIPOQ protein in serum during postnatal development of mice. ( a ) Genotypes. ( b ) mRNA levels of Adipoq and genes related to fatty acid synthesis ( Albp , Fas , Accα and Accβ ) and fatty acid oxidation ( Aco ). ( c ) Serum ADIPOQ concentration. The data shown are means ± SEM for 5–11 mice per group in a single experiment. * P < 0.05, ** P < 0.01 versus the corresponding control cells by Student’s t -test.

Article Snippet: For preparation of BRD4-overexpressing cells, a mouse Brd4 cDNA (Unigene ID Mm.

Techniques: Gene Expression, Concentration Assay, Control