Journal: Cardiovascular Therapeutics

Article Title: Epigenetic Modulation by Apabetalone Counters Cytokine-Driven Acute Phase Response In Vitro , in Mice and in Patients with Cardiovascular Disease

doi: 10.1155/2020/9397109

Figure Lengend Snippet: BET proteins contribute to cytokine-mediated induction of the CRP gene. CRP gene expression changes in HepaRG™ cells (a) and human primary hepatocytes (b) measured by rtPCR in response to IL-6, IL-1 β , or combined (dual) cytokine treatment (2 h). Apabetalone pretreatment (1 h, 25 μ M) counters mRNA induction in response to single or dual cytokine treatment. Gene expression is graphed relative to DMSO-treated cells. Representative data from three independent repeats is shown. Data is presented as a mean ± S.D. (c) MZ-1 degrades BRD2, BRD3, and BRD4 protein in HepaRG™ cells in a dose-dependent manner. Representative Western blot data is shown. (d) Quantification of BRD2, BRD3, and BRD4 protein bands relative to β -actin. Data is presented as the mean of four independent replicates ± S.D. (e) BET degradation by MZ-1 significantly repressed dual cytokine-induced CRP transcription. Representative data from three independent repeats is shown. Data is presented as the mean ± S.D. (f, g) Dual cytokine stimulation (2 h) increases BRD2 (f) and BRD4 (g) occupancy on the CRP transcription start site (CRP TSS) but not in a BET protein-lacking region (desert) as determined by ChIP. Pretreatment (1 h) with apabetalone (25 μ M) or JQ1 (0.5 μ M) reduces BRD4 association with the CRP promoter (g). Samples were processed in triplicate. Data is presented as the mean ± S.D. Statistical significance was determined through 1-way ANOVA followed by Tukey's multiple comparison test, where ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ns means no significant difference.

Article Snippet: Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies against BRD2, BRD3, or BRD4 (Bethyl), followed by HRP - coupled secondary antibodies (Calbiochem). β -Actin was detected with antibodies coupled to HRP (Sigma-Aldrich).

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Comparison

Journal: Redox Biology

Article Title: Brd4 inhibition attenuates unilateral ureteral obstruction-induced fibrosis by blocking TGF-β-mediated Nox4 expression

doi: 10.1016/j.redox.2016.12.031

Figure Lengend Snippet: Brd4 was up-regulated in the kidney after unilateral ureteral obstruction (UUO). (A) Brd4 mRNA levels were detected by real-time RT-PCR at day 1 and 7 after UUO. (B) Brd4 protein levels were detected by western blot analysis at day 1 and 7 after UUO. (C) Immunohistochemical staining of Brd4 in renal sections at day 1 and 7 after UUO. Values are expressed as the mean±SEM. * P <0.05, relative to the sham group, n=3.

Article Snippet: The rabbit anti-Brd4 antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining

Journal: Redox Biology

Article Title: Brd4 inhibition attenuates unilateral ureteral obstruction-induced fibrosis by blocking TGF-β-mediated Nox4 expression

doi: 10.1016/j.redox.2016.12.031

Figure Lengend Snippet: Brd4 inhibition attenuated TGF-β1-induced fibrosis and oxidative stress in HK-2 cells. (A-C) HK-2 cells were pretreated with or without JQ1 at different doses (0.1, 0.2, 0.5 μM) for 1 h, and then treated with TGF-β1 (10 ng/mL) for 24 h. (A) Real-time PCR analyses for the mRNA expression of collagen IV, α-SMA and fibronectin. * P <0.05 versus the TGF-β1 group. Bar graphs represent three independent experiments, each performed in triplicates. (B) Western blot analyses for the protein expression of collagen IV, α-SMA and fibronectin. (C) Protein levels were quantified by densitometry and normalized to the expression of GAPDH from three independent experiments. * P <0.05 versus the TGF-β1 group. (D-H) HK-2 cells were transfected with an siRNA against Brd4 or a negative control siRNA (si-NC) for 48 h or pretreated with 0.5 μM JQ1 for 1 h, and then treated with TGF-β1 (10 ng/mL) for 24 h. (D) Real-time PCR analyses for the mRNA expression of collagen IV, α-SMA and fibronectin in the indicated group. Bar graphs represent three independent experiments, each performed in triplicates. * P <0.05 versus TGF-β1; # P <0.05 versus si-NC. (E) Western blot analyses for the protein expression of collagen IV, α-SMA, and fibronectin in the indicated group. (F) Protein levels were quantified by densitometry and normalized to the expression of GAPDH from three independent experiments. * P <0.05 versus TGF-β1; # P <0.05 versus si-NC. (G) H 2 O 2 production in HK-2 cells in the indicated groups. Bar graphs represent three independent experiments, each performed in triplicates. * P <0.05 versus TGF-β1; # P <0.05 versus si-NC. (H) Representative images showing ROS stained with DCFH-DA dye in the indicated groups from three independent experiments.

Article Snippet: The rabbit anti-Brd4 antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Negative Control, Staining

Journal: Redox Biology

Article Title: Brd4 inhibition attenuates unilateral ureteral obstruction-induced fibrosis by blocking TGF-β-mediated Nox4 expression

doi: 10.1016/j.redox.2016.12.031

Figure Lengend Snippet: Brd4 inhibition blocked Nox4-mediated ROS and fibrosis in HK-2 cells. (A) Representative bands of Western blot analyses for the expression of Nox4, collagen IV, α-SMA, and fibronectin in the presence of TGF-β1 or siRNA against Nox4 from three independent experiments. (B) H 2 O 2 production measured by Amplex Red in HK-2 cells transfected with siRNA against Nox4 or a negative control siRNA in the presence or absence of TGF-β1. Bar graphs represent three independent experiments, each performed in triplicates. * P <0.05 versus si-NC. (C) Real-time PCR analyses for the mRNA expression of Nox4 under the condition indicated from three independent experiments, each performed in triplicates. * P <0.05 versus TGF-β1; # P <0.05 versus si-NC. (D) Western Blot analyses for the protein expression of Nox4 and bar graph quantification as indicated from three independent experiments. * P <0.05 versus TGF-β1; # P <0.05 versus si-NC. (E-F) HK-2 cells were treated with TGF-β1 (10 ng/mL) for 24 h in the presence or absence of JQ1. TGF-β1+JQ1-treated HK-2 cells were then infected with adenovirus carrying the human Nox4 for 48 h. (E) Representative Western blot analyses of collagen IV, α-SMA and fibronectin in the indicated groups. (F) H 2 O 2 production measured by Amplex Red in HK-2 cells in the indicated groups. Bar graphs represent three independent experiments, each performed in triplicates. * P <0.05 versus the TGF-β1+JQ1 group.

Article Snippet: The rabbit anti-Brd4 antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Inhibition, Western Blot, Expressing, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Infection

Journal: Redox Biology

Article Title: Brd4 inhibition attenuates unilateral ureteral obstruction-induced fibrosis by blocking TGF-β-mediated Nox4 expression

doi: 10.1016/j.redox.2016.12.031

Figure Lengend Snippet: Brd4 regulated Nox4 expression via the Smad and ERK pathways. (A) Western blot analyses for the protein expression of Smad3 and phosphorylated Smad3 in the indicated groups and quantitative analysis of Smad3 phosphorylation. * P <0.05 versus TGF-β1, # P <0.05 versus si-NC. (B) Western blot analyses for the protein expression of ERK1/2 and phosphorylated ERK1/2 in the indicated groups and quantitative analysis of ERK1/2 phosphorylation. * P <0.05 versus TGF-β1, # P <0.05 versus si-NC. (C) HK-2 cells were pretreated with either JQ1 (0.5 μM), SIS3 (Smad3 inhibitor, 10 μM) or U0126 (ERK1/2 inhibitor,10 μM) for 1 h and then treated with TGF-β1 (10 ng/mL) for 24 h. Western blot analyses for the protein expression of Nox4 in the indicated groups and quantification. * P <0.05 versus TGF-β1. (A-C) Each Western blot analysis is from three independent experiments. (D) Luciferase assay of Nox4 promoter activity in the presence of JQ1 or Brd4 knockdown with siRNA from three independent experiments, each performed in six replicates. * P <0.05 versus TGF-β1, # P <0.05 versus si-NC.

Article Snippet: The rabbit anti-Brd4 antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Phospho-proteomics, Luciferase, Activity Assay, Knockdown

Journal: STAR Protocols

Article Title: Cloning BRD4 long isoform into overexpression vectors for stable overexpression of BRD4-L in mammalian cells

doi: 10.1016/j.xpro.2022.101785

Figure Lengend Snippet:

Article Snippet: Download the appropriate sequences. a. Download the Reference Sequence for Homo sapiens bromodomain containing 4 (BRD4), transcript variant long, mRNA (GenBank: NM_058243.3) from the NCBI’s website, using the GenBank data base ( BRD4-L RefSeq ). b. Download the sequence for LentiV_Blast (Addgene, cat# 111887) from Addgene’s website ( LentiV_Blast ).

Techniques: Virus, Recombinant, Cloning, Gel Extraction, Plasmid Preparation, Sequencing, Over Expression, Expressing, Software, Imaging

Journal: Journal of translational medicine

Article Title: Innovative evaluation of selinexor and JQ1 synergy in leukemia therapy via C-MYC inhibition.

doi: 10.1186/s12967-025-06525-z

Figure Lengend Snippet: Fig. 3 Selinexor and JQ1 regulated the expression of C-MYC. (A-B) Expression levels of C-MYC at different concentrations of Selinexor and JQ1 in AML cells, respectively in cells MV4-11 and THP-1 at 24 h (A) and 48 h (B). (C) Detect the IC50 concentration of Selinexor in MV4-11 cells with scramble or BRD4- SgRNA. (D) Cell viability of MV4-11 cells with scramble or BRD4-SgRNA treated with different doses of Selinexor. (E) Expression levels of BRD4 and C-MYC proteins in MV4-11 cells with scramble or BRD4-SgRNA. (F) Detect the IC50 concentration of Selinexor in cells THP-1 cells with scramble or BRD4-SgRNA. (G) Cell viability of THP-1 cells with scramble or BRD4-SgRNA treated with different doses of Selinexor. (H) Expression levels of BRD4 and C-MYC proteins in THP-1 cells with scramble or BRD4-SgRNA. (I) Protein expression levels of C-MYC in MOLM13 cells transfected with vector or overexpressed C-MYC plasmid. (J) Cell viability of MOLM13 cells with scramble or C-MYC treated with different doses of Selinexor and JQ1. (K-L) RNA expression of C-MYC in MV4-11 (K) and MOLM13 (L) cells treated with different doses of Selinexor(50nM) and JQ1(50nM). All experiments were performed with at least three independent replicates

Article Snippet: LentiGuide-Puro plasmid targeting BRD4 and packaging vector pSPAX2 (12260, Addgene), pMD2G (12259, Addgene) were cotransfected into 293T cells to produce sgRNA lentivirus.

Techniques: Expressing, Concentration Assay, Transfection, Plasmid Preparation, RNA Expression

Journal: Scientific Reports

Article Title: Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

doi: 10.1038/s41598-019-39543-1

Figure Lengend Snippet: Recruitment of XRCC1 and XRCC1-P1/3 mutant to sites of micro-irradiation damage. ( a ) Time course of XRCC1 DNA damage localization 20–120 seconds post-irradiation. ( b ) Relative levels of XRCC1 and XRCC1-P1/3 mutant localization over 120 seconds post-irradiation. ( c ) Time course of XRCC1 DNA damage localization 2–30 min post-irradiation. ( d ) Comparison of number of cells showing detectable localization of XRCC1 at damage sites over 62 min following irradiation. Blue and orange bars represent wild type and P1/3 mutant XRCC1, respectively. Scale bars, 5 µm.

Article Snippet: Primers used in PCR reactions for vector construction are listed in Supplementary Fig. . PCR products were first moved into a pDONR201 entry vector (Invitrogen) and subsequently recombined into destination vectors, pDEST15 (Invitrogen) for full length XRCC1; pDEST544 (Addgene 11519) for XRCC1 219–415 and XRCC1 219–300 ; and pDEST17 (Invitrogen) for XRCC1 1–183 and XRCC1 301–415 .

Techniques: Mutagenesis, Irradiation, Comparison

Journal: Scientific Reports

Article Title: Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

doi: 10.1038/s41598-019-39543-1

Figure Lengend Snippet: Comparison of DNA binding activities of XRCC1 truncations. ( a ) Domain organization of XRCC1 with truncation boundaries indicated by arrows: Full length (blue), 1–183 (purple), 219–633 (green), 219–415 (red), 219–300 (orange) and 301–415 (cyan). ( b – g ) DNA binding activity of XRCC1 truncations (µM concentrations) monitored by electrophoretic mobility shift using fluorescent 39 bp duplex DNA. Discontinuous PAGE was used to facilitate band resolution. Full length gels are in Supplementary Fig. .

Article Snippet: Primers used in PCR reactions for vector construction are listed in Supplementary Fig. . PCR products were first moved into a pDONR201 entry vector (Invitrogen) and subsequently recombined into destination vectors, pDEST15 (Invitrogen) for full length XRCC1; pDEST544 (Addgene 11519) for XRCC1 219–415 and XRCC1 219–300 ; and pDEST17 (Invitrogen) for XRCC1 1–183 and XRCC1 301–415 .

Techniques: Comparison, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay

Journal: Scientific Reports

Article Title: Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

doi: 10.1038/s41598-019-39543-1

Figure Lengend Snippet: Comparison of XRCC1-CDB binding affinities for different DNA substrates. ( a ) Schematic of DNA substrates analyzed for XRCC1-CDB interaction. All substrates contained a 3′-hydroxyl group at the site of damage. Substrates that contained a 5′-phosphate group at the damage site are labelled. ( b ) Comparison of binding curves of XRCC1-CDB with each substrate showed only minor differences in affinity.

Article Snippet: Primers used in PCR reactions for vector construction are listed in Supplementary Fig. . PCR products were first moved into a pDONR201 entry vector (Invitrogen) and subsequently recombined into destination vectors, pDEST15 (Invitrogen) for full length XRCC1; pDEST544 (Addgene 11519) for XRCC1 219–415 and XRCC1 219–300 ; and pDEST17 (Invitrogen) for XRCC1 1–183 and XRCC1 301–415 .

Techniques: Comparison, Binding Assay

Journal: Scientific Reports

Article Title: Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

doi: 10.1038/s41598-019-39543-1

Figure Lengend Snippet: Small angle X-ray scattering of the XRCC1-CDB DNA-bound complex. Pair distribution curves (left) and resulting molecular envelopes (right) of ( a ) 39 bp DNA and ( b ) XRCC1-CDB with the estimated dimensions given in angstroms. An ab initio model for XRCC1-CDB was generated with DAMMIF (grey model) with subsequent BUNCH modelling (purple) to populate atoms not present in the BRCT1 domain determined by NMR (PDB 2D8M). ( c ) The pair distribution function for the XRCC1-CDB DNA-bound complex is shown in the left side of the panel. A corresponding MONSA generated model is shown in stereo. Purple spheres correspond to XRCC1-CDB while the yellow spheres correspond to 39 bp DNA.

Article Snippet: Primers used in PCR reactions for vector construction are listed in Supplementary Fig. . PCR products were first moved into a pDONR201 entry vector (Invitrogen) and subsequently recombined into destination vectors, pDEST15 (Invitrogen) for full length XRCC1; pDEST544 (Addgene 11519) for XRCC1 219–415 and XRCC1 219–300 ; and pDEST17 (Invitrogen) for XRCC1 1–183 and XRCC1 301–415 .

Techniques: Generated

Journal: Scientific Reports

Article Title: Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

doi: 10.1038/s41598-019-39543-1

Figure Lengend Snippet: Comparison of DNA Binding affinity for XRCC1-CDB mutants. ( a ) Sequence of the XRCC1 N-terminal linker. Positively charged residues that were targeted for alanine substitution are highlighted in bold. ( b ) Binding curves generated from EMSA analysis for each alanine substituted mutant (right) and the corresponding K d values (left). ( c ) Sequence alignment of XRCC1 from human, hamster, frog and Arabidopsis (plant). Conserved positively charged residues, green; glycine and proline, cyan; negatively charged residues, pink; and hydrophobic residues, yellow. ( d ) A comparison of DNA binding levels for mutant and wild type XRCC1 at 2 µM protein concentration. Each experiment was repeated three times. Mutant P1/3 had no measurable DNA binding activity at this concentration.

Article Snippet: Primers used in PCR reactions for vector construction are listed in Supplementary Fig. . PCR products were first moved into a pDONR201 entry vector (Invitrogen) and subsequently recombined into destination vectors, pDEST15 (Invitrogen) for full length XRCC1; pDEST544 (Addgene 11519) for XRCC1 219–415 and XRCC1 219–300 ; and pDEST17 (Invitrogen) for XRCC1 1–183 and XRCC1 301–415 .

Techniques: Comparison, Binding Assay, Sequencing, Generated, Mutagenesis, Protein Concentration, Activity Assay, Concentration Assay

Journal: Scientific Reports

Article Title: Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

doi: 10.1038/s41598-019-39543-1

Figure Lengend Snippet: Comparison of XRCC1 foci formation following 10 mM H2O2 treatment. ( a ) Cells expressing wild type XRCC1 (left) or P1/3 variant (right). DNA, stained with DAPI (blue colour); XRCC1 fused with YFP (green). One single confocal image taken at the center of the nucleus is presented. ( b ) 3D-stacks were acquired and visualized with Volume Viewer. The colour code reflects the position of the foci in the 3D space. ( c ) The foci number were counted with the ImageJ plugin 3D Object Counter. Results for 7 representative cells expressing each of the XRCC1 variants are displayed in the scatter dot plot. Graph representation and Mann-Whitney statistical test were performed with GraphPad Prism version 7, 02. Scale bars, 5 µm.

Article Snippet: Primers used in PCR reactions for vector construction are listed in Supplementary Fig. . PCR products were first moved into a pDONR201 entry vector (Invitrogen) and subsequently recombined into destination vectors, pDEST15 (Invitrogen) for full length XRCC1; pDEST544 (Addgene 11519) for XRCC1 219–415 and XRCC1 219–300 ; and pDEST17 (Invitrogen) for XRCC1 1–183 and XRCC1 301–415 .

Techniques: Comparison, Expressing, Variant Assay, Staining, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Identification of an XRCC1 DNA binding activity essential for retention at sites of DNA damage

doi: 10.1038/s41598-019-39543-1

Figure Lengend Snippet: Effect of P1/3 mutation on DNA repair of single strand breaks. Relative repair efficiency of single strand breaks compared for CHO cells containing an XRCC1 knock out and the same cells complemented with either WT or P1/3 mutant XRCC1. The average tail moments are shown for each cell line after 30 min of recovery following initial damage with 10 mM hydrogen peroxide. Representative images from comet assays, which were used for average tail moment calculation, are provided in the lower panels.

Article Snippet: Primers used in PCR reactions for vector construction are listed in Supplementary Fig. . PCR products were first moved into a pDONR201 entry vector (Invitrogen) and subsequently recombined into destination vectors, pDEST15 (Invitrogen) for full length XRCC1; pDEST544 (Addgene 11519) for XRCC1 219–415 and XRCC1 219–300 ; and pDEST17 (Invitrogen) for XRCC1 1–183 and XRCC1 301–415 .

Techniques: Mutagenesis, Knock-Out