Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Binding affinities and stoichiometry of binding (N) obtained via isothermal titration calorimetry (ITC) for acetylated histone peptide binding to the ATAD2 bromodomain (BRD).

Article Snippet: We recently identified eleven post-translationally modified histone ligands recognized by the ATAD2 bromodomain using the dCypher array technology developed by EpiCypher [ ].

Techniques: Binding Assay, Isothermal Titration Calorimetry, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Isothermal titration calorimetry (ITC) measurements for the ATAD2 bromodomain interaction with the histone tail ligands. ( A – K ) Exothermic ITC enthalpy plots for binding of ATAD2 bromodomain with mono-, and di-acetylated histone ligands. Calculated binding constants and peptides are indicated for each trace.

Article Snippet: We recently identified eleven post-translationally modified histone ligands recognized by the ATAD2 bromodomain using the dCypher array technology developed by EpiCypher [ ].

Techniques: Isothermal Titration Calorimetry, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Chemical shift perturbation of the ATAD2 bromodomain (BRD) residues upon interaction with histone H4 ligands. ( A ) Histogram showing the normalized 1 H, 15 N chemical shift changes in the assigned backbone amides of the ATAD2 BRD upon addition of H4K5ac (1–10), H4K8ac (1–10), and H4K5acK8ac (1–10) histone peptides in a 1:5 protein to peptide, molar ratio. ( B ) Histogram showing the normalized 1 H, 15 N chemical shift changes in the assigned backbone amides of the ATAD2 BRD upon addition of K4K5ac (1–15), H4K12ac (1–15) and H4K5acK12ac (1–15) histone peptides in a 1:5 molar ratio of protein to peptide. Residues highlighted in red correspond to the “RVF shelf” motif and the conserved asparagine 1064. Residues highlighted by the asterisk (*) denote prolines. The gaps in the residue axis denote unassigned amino acids.

Article Snippet: We recently identified eleven post-translationally modified histone ligands recognized by the ATAD2 bromodomain using the dCypher array technology developed by EpiCypher [ ].

Techniques: Residue

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Chemical shift mapping of the ATAD2 bromodomain (BRD) highlighting the histone binding pocket. Chemical shift changes induced by addition of 1:5 molar ratio of BRD to peptides are mapped onto the crystal structure of the apo ATAD2 BRD (PDB ID: 3DAI). Residues that are not assigned are colored grey, residues exhibiting 1 or 2 standard deviations from the average chemical shift change are colored orange and red, respectively. ( A ) Perturbations caused by addition of mono acetylated H4K5ac (1–10) histone peptide. ( B ) Perturbations caused by addition of di-acetylated H4K5acK8ac (1–10) histone peptide. ( C ) Perturbations caused by addition of mono-acetylated H4K5ac (1–15) histone peptide and ( D ) Perturbations caused by addition of mono-acetylated H4K5acK12ac (1–15) histone peptide.

Article Snippet: We recently identified eleven post-translationally modified histone ligands recognized by the ATAD2 bromodomain using the dCypher array technology developed by EpiCypher [ ].

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Molecular weight distribution of apo ATAD2 bromodomain (BRD) and in complex with histone H4 peptides. The molecular weights of the ATAD2 bromodomain by itself (black) and in complex with a 1:1 molar ratio with H4 unmodified (4–17) (dark blue), H4K5ac (1–15) (yellow), H4K8ac (1–10) (red), H4K12ac (1–15) (green), H4K5acK8ac (1–10) (orange) and H4K5acK12ac (1–15) (light blue) histone peptides. All molar masses are consistent with a 1:1 ratio of ATAD2 with one peptide molecule.

Article Snippet: We recently identified eleven post-translationally modified histone ligands recognized by the ATAD2 bromodomain using the dCypher array technology developed by EpiCypher [ ].

Techniques: Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: 15 N, T 1 and T 2 relaxation rate estimates for ATAD2 bromodomain (BRD) alone and in complex with histone H4K5acK12ac (1–15) peptide.

Article Snippet: We recently identified eleven post-translationally modified histone ligands recognized by the ATAD2 bromodomain using the dCypher array technology developed by EpiCypher [ ].

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Binding affinities and stoichiometry of binding (N) obtained via ITC for acetylated histone H4 peptides binding to the ATAD2 bromodomain (residues 966–1112, C1101A).

Article Snippet: We recently identified eleven post-translationally modified histone ligands recognized by the ATAD2 bromodomain using the dCypher array technology developed by EpiCypher [ ].

Techniques: Binding Assay, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Data collection and refinement statistics for the crystal structure of the ATAD2 bromodomain (BRD) bound to histone H4K5acK8ac (1–10).

Article Snippet: We recently identified eleven post-translationally modified histone ligands recognized by the ATAD2 bromodomain using the dCypher array technology developed by EpiCypher [ ].

Techniques: Solvent

Journal: International Journal of Molecular Sciences

Article Title: Coordination of Di-Acetylated Histone Ligands by the ATAD2 Bromodomain

doi: 10.3390/ijms22179128

Figure Lengend Snippet: Coordination of the H4K5acK8ac (1–10) histone ligand by the ATAD2 bromodomain (BRD). ( A ) Ligand coordination for ATAD2 BRD (cyan) with H4K5acK8ac (1–10) (green) (PDBID: 7M98). Hydrogen bonds are indicated as dashed red lines. ( B ) LigPlot + was used to depict coordination of the H4K5ac (1–10) ligand (green) by the ATAD2 BRD. Five polar contacts to D1020, Y1063, N1064, and R1067 are displayed, as well as the hydrophobic interactions assisting in ligand coordination. ( C ) Electron density for the simulated annealing composite omit map (blue) contoured at 1 s for the histone H4K5acK8ac (1–10) ligand (green). Clear electron density is observed for the first five amino acids including the K5ac sidechain. ( D ) Alignment of the ATAD2 BRD structures 4TT2 (magenta), 4QUU (salmon), and 7M98 (cyan). Highlighted residues are labeled in the protein backbone. The first three ligand residues display a different coordination between the overlaid structures and are labeled. Figures were generated with the PyMOL Molecular Graphics System, version 2.3, Schrödinger, LLC .

Article Snippet: We recently identified eleven post-translationally modified histone ligands recognized by the ATAD2 bromodomain using the dCypher array technology developed by EpiCypher [ ].

Techniques: Labeling, Generated

Journal: American Journal of Translational Research

Article Title: AGAP2-AS1/BRD7/c-Myc signaling axis promotes skin cutaneous melanoma progression

doi:

Figure Lengend Snippet: AGAP2 Antisense RNA 1 (AGAP2-AS1) interacts with bromodomain containing 7 (BRD7) in skin cutaneous melanoma (SKCM) cells. A. Flowchart of RNA pull-down and mass spectrometry assays. B. Venn diagrams of proteins identified using mass spectrometry, from three independent RNA pull-down assays. C. Biotinylated AGAP2-AS1 RNAs were incubated with A875 and A375 cell lysates, respectively. RNA-protein complexes were examined using western blot analysis with anti-BRD7 antibody. The antisense strand of AGAP2-AS1 was used as the negative control (NC). D, E. RIP experiments of AGAP2-AS1 binding to BRD7 in A875 and A375 cell lysates. n = 3 for each group. Rabbit IgG was included as the NC for the immunoprecipitation.

Article Snippet: After lysis (cell lysis buffer) and centrifugation (12,000×g, 15 min), the supernatant was incubated with anti-BRD7 antibody (ProteinTech) (4°C, overnight).

Techniques: Mass Spectrometry, Incubation, Western Blot, Negative Control, Binding Assay, Immunoprecipitation

Journal: American Journal of Translational Research

Article Title: AGAP2-AS1/BRD7/c-Myc signaling axis promotes skin cutaneous melanoma progression

doi:

Figure Lengend Snippet: Bromodomain containing 7 (BRD7) knockdown suppressed proliferation and migration of skin cutaneous melanoma (SKCM) cells. A. A875 and A375 cells transfected with si-NC (negative control) or si-BRD7#1 and si-BRD7#2 were subjected to quantitative real-time polymerase chain reaction (qRT-PCR) for BRD7 expression. n = 3 for each group. B, C. Cell Counting Kit 8 (CCK-8) assay of proliferation ability in transfected cells. n = 4 for each group. D, E. Colony formation assay of cloning ability in transfected cells. n = 3 for each group. F, G. Transwell assay of migration in transfected cells. n = 3 for each group. Scale bar: 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: After lysis (cell lysis buffer) and centrifugation (12,000×g, 15 min), the supernatant was incubated with anti-BRD7 antibody (ProteinTech) (4°C, overnight).

Techniques: Knockdown, Migration, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Cell Counting, CCK-8 Assay, Colony Assay, Cloning, Transwell Assay

Journal: American Journal of Translational Research

Article Title: AGAP2-AS1/BRD7/c-Myc signaling axis promotes skin cutaneous melanoma progression

doi:

Figure Lengend Snippet: C-Myc is a potential downstream target of the AGAP2-AS1/BRD7 axis. A-D. Western blot analysis of c-Myc after AGAP2 Antisense RNA 1 (AGAP2-AS1) or bromodomain containing 7 (BRD7) downregulation or overexpression in A875 and A375 cells. Tubulin was used as an internal control. n = 3 for each group. E, F. Western blot analysis of BRD7 after AGAP2-AS1 downregulation in A875 and A375 cells. n = 3 for each group. G. Co-immunoprecipitation assay of interaction between BRD7 and c-Myc in A875 and A375 cells transfected with si-AGAP2-AS1 or si-NC (negative control). *P < 0.05, ***P < 0.001; n.s., not significant.

Article Snippet: After lysis (cell lysis buffer) and centrifugation (12,000×g, 15 min), the supernatant was incubated with anti-BRD7 antibody (ProteinTech) (4°C, overnight).

Techniques: Western Blot, Over Expression, Control, Co-Immunoprecipitation Assay, Transfection, Negative Control

Journal: American Journal of Translational Research

Article Title: AGAP2-AS1/BRD7/c-Myc signaling axis promotes skin cutaneous melanoma progression

doi:

Figure Lengend Snippet: Regulation of c-Myc is involved in the oncogene function of AGAP2 Antisense RNA 1 (AGAP2-AS1). (A, B) Cell Counting Kit 8 (CCK-8) and (C, D) colony formation assays to determine cell viability of A875 and A375 cells co-transfected with pcDNA3.1-MYC and si-AGAP2-AS1 or si-BRD7. (E, F) Transwell assays to investigate changes in migratory abilities of A875 and A375 cells co-transfected with pcDNA3.1-MYC and si-AGAP2-AS1 or si-BRD7. Scale bar: 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: After lysis (cell lysis buffer) and centrifugation (12,000×g, 15 min), the supernatant was incubated with anti-BRD7 antibody (ProteinTech) (4°C, overnight).

Techniques: Cell Counting, CCK-8 Assay, Transfection

Journal: Frontiers in Oncology

Article Title: The MYC Paralog-PARP1 Axis as a Potential Therapeutic Target in MYC Paralog-Activated Small Cell Lung Cancer

doi: 10.3389/fonc.2020.565820

Figure Lengend Snippet: Effects of JQ1 and BMN673 on the HR repair pathway. (A) Representative images of Rad51 immunofluorescence staining in SCLC cells treated with drugs as indicated for 24 h. Scale bar, 20 μm. Quantification of Rad51 fluorescence intensities from three independent experiments was shown as mean ± S.D. **P < 0.01; ***P < 0.001. (B) Western blot analysis of Rad51 expression in SCLC cells treated with drugs as indicated for 24 h. (C) Western blot analysis of indicated proteins in DMS273 cell upon knockdown of BRD2 or BRD3 or BRD4 . (D) ChIP-PCR analysis displays the decreased association of BRD4 in the promoter of RAD51 in DMS273 and H526 cells treatment with JQ1 for 24 h. *P < 0.05; **P < 0.01; ns, no significance. (E) Western blot analysis of phosphorylated-DNA-PKcs and phosphorylated-RPA32 in MYC paralog-dependent and independent-SCLC cells treated with indicated drugs for 24 h. β-actin was used as a loading control.

Article Snippet: Antibodies against the following proteins were used for our studies: c-MYC (1:1,000, abcam ab32072), PARP1 (1:1,000, Affinity DF7198), GAPDH (1:10,000, Affinity AF7021), Rad51 (1:10,000, Abcam ab133534), PARP (1:1,000, CST 9542), γH2AX (1:1,000, CST 2577), p-CHK1 (1:1,000, Ser317, CST 12302), BRD2 (1:1,000, Proteintech 22236-1-AP), BRD3 (1:1,000, Proteintech 11859-1-AP), BRD4 (1:500, Bethyl A301-985A50), p-DNA-PKcs (1:1,000, Abcam ab124918), p-RPA32 (1:5,000, Ser4/Ser8, Novus), MYCN (1:1,000, CST 9405), MYCL (1:1,000 R&D, AF4050) and β-actin (1:10,000, Transgen HC201-02).

Techniques: Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing, Knockdown, Control

Journal: Cell death & disease

Article Title: The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells.

doi: 10.1038/s41419-023-06273-2

Figure Lengend Snippet: Fig. 1 BRD4 inhibition protects osteosarcoma cells from erastin-ferrop in vitro. A Relative protein expression levels of BRD4 tested by western blotting in SaoS2 and U2-OS cells. B Cell survival rate analysis through the MTT assay. Intracellular MDA (C) and Fe2+ (D) content tested by analytical kits. E lipid ROS of each group determined by the boron-dipyrromethene C-11 probe and flow cytometry. F Electron microscopic images of mitochondria and the percentage of the damaged mitochondria. Tukey–Kramer test of one-way ANOVA, *: P < 0.05; **: P < 0.01; ***: P < 0.005.

Article Snippet: Then, primary antibodies of BRD4 (Merck KGaA, Cat. No. PLA0227), ACSL3 (Merck KGaA, Cat. No. HPA071021), ACSL4 (Proteintech, Cat. No. 22401-1-AP), ACSL5 (Proteintech, Cat No. 15708-1-AP), ACSL6 (Abcam, Cat. No. ab229937), SRPK2 (Abcam, Cat. No. ab251113), p-SRPK2 (CST, Cat. No. 23708), mTOR (CST, Cat. No. 2983), p-mTOR (CST, Cat. No. 5536), p70 S6K (CST, Cat. No. 9202), p-p70 S6K (CST, Cat. No. 97596), and SRSF2 (Merck KGaA, Cat. No. HPA049905) were diluted to a working concentration and incubated overnight at 4 °C.

Techniques: Inhibition, In Vitro, Expressing, Western Blot, MTT Assay, Cytometry

Journal: Cell death & disease

Article Title: The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells.

doi: 10.1038/s41419-023-06273-2

Figure Lengend Snippet: Fig. 2 BRD4 inhibition protects osteosarcoma cells from erastin-ferrop in vivo. A Flowchart guide for the animal experiments. B Subcutaneous tumors from individual mice. Growth curve (C) and weights (D) of subcutaneous tumors. E MDA content in tumors. F Prussian blue staining of ferric ions and probe hybridization of lipid ROS in tumor tissue slides. Dunnett’s test of one-way ANOVA, *: P < 0.05; **: P < 0.01; ***: P < 0.005; n.s.: no significance.

Article Snippet: Then, primary antibodies of BRD4 (Merck KGaA, Cat. No. PLA0227), ACSL3 (Merck KGaA, Cat. No. HPA071021), ACSL4 (Proteintech, Cat. No. 22401-1-AP), ACSL5 (Proteintech, Cat No. 15708-1-AP), ACSL6 (Abcam, Cat. No. ab229937), SRPK2 (Abcam, Cat. No. ab251113), p-SRPK2 (CST, Cat. No. 23708), mTOR (CST, Cat. No. 2983), p-mTOR (CST, Cat. No. 5536), p70 S6K (CST, Cat. No. 9202), p-p70 S6K (CST, Cat. No. 97596), and SRSF2 (Merck KGaA, Cat. No. HPA049905) were diluted to a working concentration and incubated overnight at 4 °C.

Techniques: Inhibition, In Vivo, Staining, Hybridization

Journal: Cell death & disease

Article Title: The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells.

doi: 10.1038/s41419-023-06273-2

Figure Lengend Snippet: Fig. 3 The dual effects of BRD4 on ACSL3 expression and subcellular location. A Functional domains of the complete ACSL3 protein with its corresponding aa length and the alignment result of the aa sequences for nine potential isoforms of ACSL3 collected from the UniProt database. B The genomic structures of ACSL3 exon skipping events of TCGA and GTEx across reference gene model from the ExonskipDB database. C, E Relative protein levels detected by western blotting. D Relative mRNA levels detected by RT-qPCR. F RNA stability assessed by RNA digestibility tests. G Detection of splicing variants of ACSL3 via RT-qPCR and agarose gel electrophoresis. Dunnett’s test of one-way ANOVA, *: P < 0.05; **: P < 0.01; ***: P < 0.005. H Typical images of ACSL3 and mitochondrial localization captured by laser confocal microscopy.

Article Snippet: Then, primary antibodies of BRD4 (Merck KGaA, Cat. No. PLA0227), ACSL3 (Merck KGaA, Cat. No. HPA071021), ACSL4 (Proteintech, Cat. No. 22401-1-AP), ACSL5 (Proteintech, Cat No. 15708-1-AP), ACSL6 (Abcam, Cat. No. ab229937), SRPK2 (Abcam, Cat. No. ab251113), p-SRPK2 (CST, Cat. No. 23708), mTOR (CST, Cat. No. 2983), p-mTOR (CST, Cat. No. 5536), p70 S6K (CST, Cat. No. 9202), p-p70 S6K (CST, Cat. No. 97596), and SRSF2 (Merck KGaA, Cat. No. HPA049905) were diluted to a working concentration and incubated overnight at 4 °C.

Techniques: Expressing, Functional Assay, Western Blot, Quantitative RT-PCR, Agarose Gel Electrophoresis, Confocal Microscopy

Journal: Cell death & disease

Article Title: The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells.

doi: 10.1038/s41419-023-06273-2

Figure Lengend Snippet: Fig. 5 The effects of BRD4 on erastin-ferrop are partly working through the ACSL3 pathway. A, G Cell survival rate. B, H Intracellular content of MDA. C, I Intracellular content of Fe2+. E Relative protein levels detected by western blotting. F Abundance analysis of intracellular arachidonic acid. D, J Positive rate of intracellular lipid ROS. Dunnett’s test of one-way ANOVA, *: P < 0.05; **: P < 0.01; ***: P < 0.005; n.s.: no significant.

Article Snippet: Then, primary antibodies of BRD4 (Merck KGaA, Cat. No. PLA0227), ACSL3 (Merck KGaA, Cat. No. HPA071021), ACSL4 (Proteintech, Cat. No. 22401-1-AP), ACSL5 (Proteintech, Cat No. 15708-1-AP), ACSL6 (Abcam, Cat. No. ab229937), SRPK2 (Abcam, Cat. No. ab251113), p-SRPK2 (CST, Cat. No. 23708), mTOR (CST, Cat. No. 2983), p-mTOR (CST, Cat. No. 5536), p70 S6K (CST, Cat. No. 9202), p-p70 S6K (CST, Cat. No. 97596), and SRSF2 (Merck KGaA, Cat. No. HPA049905) were diluted to a working concentration and incubated overnight at 4 °C.

Techniques: Western Blot

Journal: Cell death & disease

Article Title: The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells.

doi: 10.1038/s41419-023-06273-2

Figure Lengend Snippet: Fig. 6 SRPK2 is recruited by BRD4 and binds to its CTD domain. A 28 splicesome-associated proteins that bind to BRD4 via Co-IP and MS assay. B Unique sequences of SRPK1 and SRPK2 that bound to BRD4 protein analyzed by MS. C Relative mRNA levels detected by RT-qPCR. Dunnett’s test of one-way ANOVA, *: P < 0.05; **: P < 0.01; ***: P < 0.005. D RNA stability assessed by RNA digestibility tests. E IF staining results of ACSL3 in cells, typical images taken with laser confocal microscopy. F Endogenous binding relationship of BRD4 and SRPK2 identified by Co-IP method. G Yeast hybrid system to verify the binding domain of BRD4 (BD1, BD2, ET, and CTD domains) to SRPK2.

Article Snippet: Then, primary antibodies of BRD4 (Merck KGaA, Cat. No. PLA0227), ACSL3 (Merck KGaA, Cat. No. HPA071021), ACSL4 (Proteintech, Cat. No. 22401-1-AP), ACSL5 (Proteintech, Cat No. 15708-1-AP), ACSL6 (Abcam, Cat. No. ab229937), SRPK2 (Abcam, Cat. No. ab251113), p-SRPK2 (CST, Cat. No. 23708), mTOR (CST, Cat. No. 2983), p-mTOR (CST, Cat. No. 5536), p70 S6K (CST, Cat. No. 9202), p-p70 S6K (CST, Cat. No. 97596), and SRSF2 (Merck KGaA, Cat. No. HPA049905) were diluted to a working concentration and incubated overnight at 4 °C.

Techniques: Co-Immunoprecipitation Assay, Quantitative RT-PCR, Staining, Confocal Microscopy, Binding Assay

Journal: Cell death & disease

Article Title: The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells.

doi: 10.1038/s41419-023-06273-2

Figure Lengend Snippet: Fig. 7 BRD4 affects splicing efficiency of pre-mACSL3 through SRPK2. A, D Relative protein levels detected by western blotting. B The enrichment of ACSL3 mRNA in the immunoprecipitate product of anti-SRSF2 antibody via the RIP/ RT-qPCR assay. C Relative mRNA levels detected by RT-qPCR. E RNA stability assessed by RNA digestibility tests. Dunnett’s test of one-way ANOVA, *: P < 0.05; **: P < 0.01; ***: P < 0.005.

Article Snippet: Then, primary antibodies of BRD4 (Merck KGaA, Cat. No. PLA0227), ACSL3 (Merck KGaA, Cat. No. HPA071021), ACSL4 (Proteintech, Cat. No. 22401-1-AP), ACSL5 (Proteintech, Cat No. 15708-1-AP), ACSL6 (Abcam, Cat. No. ab229937), SRPK2 (Abcam, Cat. No. ab251113), p-SRPK2 (CST, Cat. No. 23708), mTOR (CST, Cat. No. 2983), p-mTOR (CST, Cat. No. 5536), p70 S6K (CST, Cat. No. 9202), p-p70 S6K (CST, Cat. No. 97596), and SRSF2 (Merck KGaA, Cat. No. HPA049905) were diluted to a working concentration and incubated overnight at 4 °C.

Techniques: Western Blot, Quantitative RT-PCR

Journal: Cell death & disease

Article Title: The BRD4-SRPK2-SRSF2 signal modulates the splicing efficiency of ACSL3 pre-mRNA and influences erastin-induced ferroptosis in osteosarcoma cells.

doi: 10.1038/s41419-023-06273-2

Figure Lengend Snippet: Fig. 8 Correlation scatter plot between BRD4, SRPK2, SRSF2 and ACSL3 in osteosarcoma tissue. A Data from the GEO database (n = 18, Bivariate correlation analysis, R ≤0.4: low correlation; R å 0.4: middle correlation; P < 0.05: significant). B Intra- patient variation in diversity index (n = 10, Wilcoxon signed-rank test). C Schematic diagram of BRD4/SRPK2/SRSF2 axis in pre-mACSL3 splicing and expression.

Article Snippet: Then, primary antibodies of BRD4 (Merck KGaA, Cat. No. PLA0227), ACSL3 (Merck KGaA, Cat. No. HPA071021), ACSL4 (Proteintech, Cat. No. 22401-1-AP), ACSL5 (Proteintech, Cat No. 15708-1-AP), ACSL6 (Abcam, Cat. No. ab229937), SRPK2 (Abcam, Cat. No. ab251113), p-SRPK2 (CST, Cat. No. 23708), mTOR (CST, Cat. No. 2983), p-mTOR (CST, Cat. No. 5536), p70 S6K (CST, Cat. No. 9202), p-p70 S6K (CST, Cat. No. 97596), and SRSF2 (Merck KGaA, Cat. No. HPA049905) were diluted to a working concentration and incubated overnight at 4 °C.

Techniques: Expressing

Journal: PLOS Pathogens

Article Title: Merkel cell polyomavirus small T antigen is a viral transcription activator that is essential for viral genome maintenance

doi: 10.1371/journal.ppat.1011039

Figure Lengend Snippet: (A) Confirmation of LTSD-dependent direct sT WT interaction with Cux1, c-Jun, CBP, and BRD9 by immunoprecipitation (IP). N-terminally SF-tagged (NSF) NSF-sT WT and NSF-sT LTSD cell lysates were immunoprecipitated with FLAG Ab. 2.5% of IP lysate was used as input. SF-sT was pulled down to confirm equal precipitation of sT WT and sT LTSD . PP2Ac, which interacts with both sT WT and sT LTSD , was used as a positive control. (B) MCVΔsT transfected 293 TRE NSF-sT WT and NSF-sT LTSD cells were treated with Dox at day 1 p.t. and harvested day 4 p.t. for immunofluorescence. Cells were co-stained with antibodies against FLAG sT (red, Alexa 488) and either Cux1, c-Jun, or CBP (green, Alexa 568), and images were captured by confocal microscopy. Magnification 100x. Arrowheads designate sT-positive mitotic cells. White bars on DAPI images indicate 10 μm.

Article Snippet: Primary antibodies include: Cux1 (11733-1-AP, Proteintech), BRD9 (24785-1-AP, Proteintech), c-Jun (24909-1-AP, Proteintech), CBP (7389S, Cell Signaling), DYKDDDDK Tag (FLAG) (A00187S, Genscript), Total H3 (14269, Cell Signaling), H3K18Ac (13998, Cell Signaling), Anti-SV40 T antigen PAb419 (sc-58665, Santa Cruz), and Hsp70 (sc-7298, Santa Cruz).

Techniques: Immunoprecipitation, Positive Control, Transfection, Immunofluorescence, Staining, Confocal Microscopy

Journal: PLOS Pathogens

Article Title: Merkel cell polyomavirus small T antigen is a viral transcription activator that is essential for viral genome maintenance

doi: 10.1371/journal.ppat.1011039

Figure Lengend Snippet: (A) Effect o f siRNA knockdown of sT interactors on viral gene expression in MCV WT . siRNA treatment of MCV WT samples. In cells treated with siCux1.1, siCux1.2, sic-Jun.1, and sic-Jun.2, early and late gene expression increases relative to the siCtrl. In cells treated with siCBP.2 and siBRD9.2, early and late gene expression decreases. Total RNA was subjected to qRT-PCR analysis by the 2 -ΔΔCt method using VP2 (set 1) and PanT primer pairs with 18S ribosomal RNA for normalization. Error bars indicate SD. Unpaired T-test was used for statistical analysis to determine the significance relative to siCtrl. *, P<0.05; **, P<0.01; ***, P<0.001. (B) A-485 and I-BRD9 treatment of MCVΔsT transfected with empty or sT. A-485 treatment significantly decreases early and late gene expression at 1.0, 5.0, and 10.0 μM in MCVΔsT transfected with sT. I-BRD9 treatment at 0.5 and 2.0 μM also significantly decreases early and late gene expression. Total extracted RNA was subjected to qRT-PCR analysis as described in Fig 8A. Relative mRNA expression to the sT WT -transfected sample is shown. Error bars indicate SD. Unpaired T-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001.

Article Snippet: Primary antibodies include: Cux1 (11733-1-AP, Proteintech), BRD9 (24785-1-AP, Proteintech), c-Jun (24909-1-AP, Proteintech), CBP (7389S, Cell Signaling), DYKDDDDK Tag (FLAG) (A00187S, Genscript), Total H3 (14269, Cell Signaling), H3K18Ac (13998, Cell Signaling), Anti-SV40 T antigen PAb419 (sc-58665, Santa Cruz), and Hsp70 (sc-7298, Santa Cruz).

Techniques: Knockdown, Gene Expression, Quantitative RT-PCR, Transfection, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Histone demethylase KDM9 activates the CCND1/AKT pathway to promote the malignant progression of gliomas through interaction with BRD2

doi: 10.1007/s00018-025-05900-9

Figure Lengend Snippet: KDM9 regulates CCND1 transcription through interaction with BRD2. ( A ) Protein silver staining of IP via KDM9 antibody enrichment in LN229 cells. ( B ) Transcription factors in the protein mass spectrometry data are shown. Exogenous and endogenous IP experiments were performed to analyze the interaction between KDM9 and BRD4 ( C ) or BRD2 ( D ). ( E ) The IF assay was used to detect the expression and co-localization of KDM9 and BRD2 in LN229 cells. ( F ) After the transfection of pcDNA3.1-BRD2-His into LN229-shKDM9 cells for 48 h, ChIP-qPCR was performed to detect the enrichment of H4K20me2 at the CCND1 P4 promoter region (with IgG as negative control). ( G ) After the transfection of wild-type or mutant KDM9 plasmids into U251 cell lines for 48 h, luciferase reporter assays were conducted to detect CCND1 transcription activity (Left). After the transfection of pcDNA3.1-BRD2-His into LN229-shKDM9 cells for 48 h, luciferase reporter assays were performed to detect CCND1 transcription activity (Right). ( H ) After the transfection of shBRD2 plasmids into U251-KDM9-WT cell lines for 48 h, luciferase reporter assays were conducted to detect CCND1 transcription activity. ( I ) After the transfection of pcDNA3.1-BRD2-His into shKDM9 glioma cells for 48 h, a Western blot experiment was conducted to detect the protein expression of KDM9, BRD2, CCND1, AKT/P-AKT, mTOR/P-mTOR, and CD133. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: CD133 (18470–1-AP, 1:1000), Histone H4 (16047–1-AP, 1:500), CCND1 (60186–1-Ig, 1:2000), SMAD2 (12570–1-AP, 1:2000), P-AKT (66444–1-IG, 1:1000), AKT (60230–2-IG, 1:1000), Flag-Tag (66008–4-Ig, 1:5000), BRD2 (22236–1-AP, 1:1000), and BRD4 (28486–1-AP, 1:1000) were purchased from Proteintech (Chicago, IL).

Techniques: Silver Staining, Mass Spectrometry, Expressing, Transfection, ChIP-qPCR, Negative Control, Mutagenesis, Luciferase, Activity Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling

doi: 10.1093/nar/gkac645

Figure Lengend Snippet: SUPT16H acetylation requires TIP60 and is recognized by BRD4. ( A ) Lysates of HEK293T, Jurkat, HeLa and NK-92 cells were incubated with an acetyl-lysine antibody or normal rabbit IgG (rIgG). Immunoprecipitated protein samples were analyzed by protein immunoblotting using SUPT16H or SSRP1 antibodies. ( B ) Lysates of HEK293T cells were incubated with a TIP60 antibody or normal mouse IgG (mIgG). Immunoprecipitated protein samples were analyzed by protein immunoblotting using SUPT16H or TIP60 antibodies. ( C ) HEK293T cells stably expressing TIP60 or non-targeting (NT) shRNA were subjected to protein immunoblotting and RT-qPCR analyses of TIP60 expression. RT-qPCR results were calculated from three independent experiments (**** P < 0.0001, Student's t -test). ( D ) Lysates of HEK293T cells in ( C ) were incubated with an acetyl-lysine antibody. Immunoprecipitated protein samples were analyzed by protein immunoblotting using a SUPT16H antibody. ( E ) Lysates of HEK293T cells treated with a TIP60-specific inhibitor MG149 were incubated with an acetyl-lysine antibody. Immunoprecipitated protein samples were analyzed by protein immunoblotting using a SUPT16H antibody. ( F ) Lysates of HEK293T cells were incubated with a BRD4 antibody (C-terminus) or rIgG. Immunoprecipitated protein samples were analyzed by protein immunoblotting using SUPT16H, SSRP1, or BRD4 (C-terminus) antibodies. ( G ) Lysates of HEK293T cells were incubated with a SUPT16H antibody or mIgG. Immunoprecipitated protein samples were analyzed by protein immunoblotting using BRD4 (C-terminus), BRD2, SSRP1 or SUPT16H antibodies. The higher band of BRD4 in ( F ) and ( G ) labeled with the asterisk was BRD4L, while the lower one was its degraded product. ( H ) Lysates of HEK293T cells treated with a BETi JQ1 (1 μM) were adjusted to assure the same input of SUPT16H, and incubated with a BRD4 antibody (C-terminus). Immunoprecipitated protein samples were analyzed by protein immunoblotting using SUPT16H or BRD4 (C-terminus) antibodies. ( I ) Lysates of HEK293T cells in ( C ) were incubated with a BRD4 antibody (C-terminus). Immunoprecipitated protein samples were analyzed by protein immunoblotting using SUPT16H or BRD4 (C-terminus) antibodies. (Int: intensity)

Article Snippet: Recombinant BRD4 bromodomain 2 (BD2) was purchased from EpiCypher.

Techniques: Incubation, Immunoprecipitation, Western Blot, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Labeling

Journal: Nucleic Acids Research

Article Title: FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling

doi: 10.1093/nar/gkac645

Figure Lengend Snippet: SUPT16H is acetylated at K674 of the middle domain (MD). ( A ) Schematic illustration of defined domains of SUPT16H protein. NTD: N-terminal domain; DD: dimerization domain; MD: middle domain; CTD: C-terminal domain. ( B ) Lysates of HEK293T cells transfected with the vector expressing indicated FLAG-tagged protein domains of SUPT16H were incubated with an acetyl-lysine or FLAG antibody, and immunoprecipitated protein samples were analyzed by protein immunoblotting using FLAG or acetyl-lysine antibodies, respectively. ( C ) HEK293T cells stably expressing TIP60 or NT shRNA were transfected with the vector expressing FLAG-tagged MD of SUPT16H. Cell lysates were incubated with acetyl-lysine or FLAG antibodies, and immunoprecipitated protein samples were analyzed by protein immunoblotting using FLAG or acetyl-lysine antibodies, respectively. ( D ) Lysates of HEK293T cells in ( C ) were incubated with a BRD4 antibody (C-terminus). Immunoprecipitated protein samples were analyzed by protein immunoblotting using FLAG or BRD4 (C-terminus) antibodies. ( E ) Prediction of acetylated lysine sites in the MD of SUPT16H by PhosphoSitePlus®. Lysine site K674 has the highest likelihood to be acetylated. ( F ) Lysates of HEK293T cells transfected with FLAG-tagged, wild-type (WT) or K674R MD of SUPT16H, were incubated with an acetyl-lysine antibody. Immunoprecipitated protein samples were analyzed by protein immunoblotting using a FLAG antibody. ( G ) Lysates of HEK293T cells in ( F ) were incubated with a BRD4 antibody (C-terminus). Immunoprecipitated protein samples were analyzed by protein immunoblotting using FLAG or BRD4 (C-terminus) antibodies.

Article Snippet: Recombinant BRD4 bromodomain 2 (BD2) was purchased from EpiCypher.

Techniques: Transfection, Plasmid Preparation, Expressing, Incubation, Immunoprecipitation, Western Blot, Stable Transfection, shRNA

Journal: Nucleic Acids Research

Article Title: FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling

doi: 10.1093/nar/gkac645

Figure Lengend Snippet: SUPT16H-BRD4 interaction prevents SUPT16H from protein degradation. ( A ) HEK293T, Jurkat, HeLa and NK-92 cells treated with JQ1 were subjected to protein immunoblotting analysis using a SUPT16H antibody. ( B ) The similar analysis as in (A) was performed for HEK293T cells treated with UMB-136 for 48 h. ( C, D ) HEK293T cells stably expressing BRD4 or NT shRNA were subjected to mRNA RT-qPCR analysis of BRD4 or SUPT16 expression ( C ) or protein immunoblotting analysis using a SUPT16H antibody (D). ( E, F ) The similar analysis as in (C, D) was performed for HEK293T cells transfected with BRD4 or NT siRNAs. Results were calculated from three independent experiments (**** P < 0.0001, Student's t test for C, one-way ANOVA for E). ( G ) Lysates of HEK293T cells treated with JQ1 (1 μM) were incubated with a SUPT16H antibody. Immunoprecipitated protein samples were analyzed by protein immunoblotting using K48-ubquitin or SUPT16H antibodies.

Article Snippet: Recombinant BRD4 bromodomain 2 (BD2) was purchased from EpiCypher.

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Transfection, Incubation, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling

doi: 10.1093/nar/gkac645

Figure Lengend Snippet: SUPT16H-BRD4 associates with HDAC1 and EZH2 contributing to gene silencing. (A, B) Lysates of HEK293T cells transfected with the vector expressing V5-tagged HDAC1 ( A ) or EZH2 ( B ) were incubated with a V5 antibody or mIgG. Immunoprecipitated protein samples were analyzed by protein immunoblotting using SUPT16H, BRD4 (C-terminus), or V5 antibodies. (C–E) Lysates of HEK293T cells were incubated with HDAC1 ( C ), EZH2 ( D ) or SUPT16H ( E ) antibodies. Immunoprecipitated protein samples were analyzed by protein immunoblotting using the indicated antibodies. (F–H) HEK293T cells treated with JQ1 ( F ), UMB-136 ( G ) or CBL0137 (500 nM) ( H ) were subjected to protein immunoblotting analysis of SUPT16H, HDAC1 and phospho-HDAC1 (ser421/423). (I–L) HEK293T cells stably expressing BRD4 or NT shRNA ( I ) were subjected to protein immunoblotting analysis of SUPT16H, HDAC1 and phospho-HDAC1 (ser421/423). The similar analysis as in ( I ) was performed for HEK293T cells transfected with BRD4 ( J ) or SUPT16H ( L ) siRNAs. Knockdown of SUPT16H by its siRNAs was confirmed by RT-qPCR to measure its mRNA level (K). ( M ) HEK293T cells treated with CBL0137 at the increasing doses (100, 200, 500 nM) were subjected to protein immunoblotting analysis using histone H3ac or total H3 antibodies. ( N, O ) Lysates of HEK293T cells transfected with SUPT16H or BRD4 siRNAs were crosslinked and incubated with HDAC1, phospho-HDAC1 (ser421/423), EZH2 antibodies, or control IgG. Immunoprecipitated DNA samples were analyzed by qPCR analysis using primers targeting the promoter region of IFI44L (N) or MX2 (O). Results were calculated from three independent experiments (**** P < 0.0001, one-way ANOVA for K, two-way ANOVA for N, O).

Article Snippet: Recombinant BRD4 bromodomain 2 (BD2) was purchased from EpiCypher.

Techniques: Transfection, Plasmid Preparation, Expressing, Incubation, Immunoprecipitation, Western Blot, Stable Transfection, shRNA, Knockdown, Quantitative RT-PCR, Control

Journal: Nucleic Acids Research

Article Title: FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling

doi: 10.1093/nar/gkac645

Figure Lengend Snippet: SUPT16H-BRD4 suppresses gene expression of IFN signaling. (A, B) HEK293T cells transfected with BRD4 ( A ) or SUPT16H ( B ) siRNAs were subjected to mRNA RT-qPCR analysis of IFNβ and ISG15 expression. ( C ) HEK293T cells transfected with SUPT16H or NT siRNAs were subjected to mRNA RT-qPCR analysis of IL-4/6/8 expression. (D, E) NK-92 cells treated with CBL0137 (500 nM) were subjected to mRNA RT-qPCR ( D ) or protein immunofluorescence ( E ) analysis of IL-4/6/8 expression. (F, G) HEK293T cells transfected with SUPT16H or NT siRNAs were further transfected with an ISRE-driven ( F ) or GAS-driven ( G ) firefly luciferase reporter plus TK-driven Renilla luciferase control vectors, which were stimulated by Type I (IFNα) or II (IFNγ) IFNs, respectively. Relative luciferase unit, RLU (firefly/ Renilla luciferase activity) from above cells was calculated and normalized to that of siNT-transfected, IFN-treated cells. (H, I) Lysates of HEK293T ( H ) or NK-92 ( I ) cells treated with CBL0137 (500 nM) were crosslinked and incubated with H3ac, H3K9me3, H3K27me3 antibodies or normal IgGs. Immunoprecipitated DNA samples were analyzed by qPCR analysis using primers targeting the promoter region of ISGs (IFI44L, MX2). Results were calculated from three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, two-way ANOVA).

Article Snippet: Recombinant BRD4 bromodomain 2 (BD2) was purchased from EpiCypher.

Techniques: Gene Expression, Transfection, Quantitative RT-PCR, Expressing, Immunofluorescence, Luciferase, Control, Activity Assay, Incubation, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: FACT subunit SUPT16H associates with BRD4 and contributes to silencing of interferon signaling

doi: 10.1093/nar/gkac645

Figure Lengend Snippet: SUPT16H–BRD4 function of gene suppression in antiviral IFN signaling. FACT subunit SUPT16H undergoes protein acetylation at K674 of MD domain, catalyzed by TIP60, which is recognized by BRD4. Such SUPT16H–BRD4 interaction prevents the protein degradation of SUPT16H. SUPT16H–BRD4 physically associates with epigenetic silencing enzymes (HDAC1, EZH2), and further affects their activation status and/or promoter association as well as modulates the histone marks (H3ac, H3K9me3, H3K27me3), which overall results in the functional overlaps of SUPT16H and BRD4 to suppress gene expression, including HERVs, non-coding repetitive elements, IFNs, and ISGs, in multiple types of cells, such as epithelial and NK cells. Furthermore, the SUPT16H inhibitor CBL0137 is potent to induce IFN signaling in both epithelial and NK cells, forming two host defense layers against viral infections.

Article Snippet: Recombinant BRD4 bromodomain 2 (BD2) was purchased from EpiCypher.

Techniques: Activation Assay, Functional Assay, Gene Expression