Journal: Advanced Science

Article Title: Epstein‐Barr Virus Expressed Long Non‐Coding RNA ( lncBART s) Regulate EBV Latent Genome Replication

doi: 10.1002/advs.202507286

Figure Lengend Snippet: Interaction of lncBART s with the N‐terminal of BRD4. a) Genome browser display of BRD4 binding to BART exons (NC_0 07605.1) based on CLIP‐seq data in C666‐1 cells. The red lines highlight the location of BRD4 binding motifs on BART exons. b) Sequence logos corresponding to enriched sequence elements identified by motif analysis of BRD4 SpyCLIP clusters. c) RNA pulldown assay using 5 µg of biotin‐labeled wild‐type RPMS1 or 5 µg of a mutant RPMS1 ( RPMS1 ‐mut) with a deletion in the BRD4 binding motif (generated by IVF) to capture 5 µg of purified BRD4 protein. In the western blot analysis, 5 µg of purified BRD4 protein was used as the input control. d) RIP analysis using BRD4‐specific or IgG control antibody was performed on C666‐1 cell extracts (n = 3), and lncBART s exons detected via RT‐qPCR. Comparisons were made between the 48 h, 500 µM JQ1‐treated group and the untreated control group. e) Representative images of RNA FISH/IF assay for lncBART s (red) and BRD4 (green) in C666‐1 cells treated with DMSO for 48h, 500 nM JQ1 for 48 h or 250 nM dBET1 for 24 h. Scale bar, 10 µm. f) Analysis of the levels of RPMS1 RNA and BRD4 protein after treatment as in d) (n = 3), measured by RT‐qPCR and western blot, respectively. Statistical analysis was performed using unpaired two‐tailed Student's t ‐test. Data are presented as mean ± SEM. ** p < 0.01, *** p < 0.001, ns, no significance.

Article Snippet: Using 5% digitonin to permeabilised the cell membranes allowedprimary antibodies against CTCF (Millipore, Cat. No 07‐729), BRD4 (EpiCypher, Cat. No 13‐2003), H3K4me3 (Epicypher, Cat. No 13‐0041), H3K27ac (Abcam, Cat. No ab4729) and IgG‐specific (Epicypher, Cat. No 13‐0042) secondary antibodies to bind to the DNA chromatin during incubation overnight at 4 °C.

Techniques: Binding Assay, Sequencing, Labeling, Mutagenesis, Generated, Purification, Western Blot, Control, Quantitative RT-PCR, FISH/IF assay, Two Tailed Test

Journal: Advanced Science

Article Title: Epstein‐Barr Virus Expressed Long Non‐Coding RNA ( lncBART s) Regulate EBV Latent Genome Replication

doi: 10.1002/advs.202507286

Figure Lengend Snippet: The BRD4/CTCF/EBNA1 complex support lncBART s’ regulatory on ori P. a) Expression levels of BRD4 were evaluated by western blot in BRD4 knockdown and control C666‐1 cells. b) Representative images of DNA FISH assay of EBV DNA copies (red) in BRD4 knockdown and control C666‐1 cells. Scale bar, 10 µm. The accompanying bar charts illustrate the quantification and statistical comparison of EBV copy number between BRD4 knockdown (n = 21) and control (n = 28) cells. c) EBV copy number as determined by qPCR in BRD4 knockdown and control C666‐1 cells (n = 3). d) Representative images of DNA FISH assay of EBV DNA copies (red) in C666‐1 cells treated with DMSO or 500 nM JQ1 for 7 days, during which the cells were passaged once. Scale bar, 10 µm. The accompanying bar charts illustrate the quantification and statistical comparison of EBV copy number between JQ1 (n = 27) and DMSO (n = 19) treated C666‐1 cells. e) EBV copy number determined by qPCR in C666‐1 cells treated with DMSO or 500 nM JQ1 for 7 days (n = 3). f) Co‐IP assays showing BRD4 interaction with CTCF and EBNA1 in lncBART s knockdown and control C666‐1 cells. Cell extracts were immunoprecipitated using a BRD4 antibody and then western blot analysis performed with antibodies against CTCF and EBNA1. RIP analysis was performed on C666‐1 cell extracts using either g) CTCF‐specific, h) EBNA1‐specific, or IgG control antibody, followed by RT‐qPCR detection of lncBART s exons. Comparisons were made between cells treated with 500 µM JQ1 for 48 h and untreated control group (n = 3). i) The CUT&RUN‐seq track images display the binding profiles of BRD4 and CTCF on the EBV genome (NC_0 07605.1) in lncBART s knockdown and control C666‐1 cells. This is visualized alongside lncBART s ChIRP (top track: RPMS1 binding) and ATAC‐seq analysis using IGV. Areas of co‐occupancy adjacent to ori P, Qp and RPMS1 revealed in ChIRP‐seq, ATAC‐seq, BRD4 and CTCF CUT&RUN‐seq are highlighted and illustrated in detail in the lower panel. Statistical analysis was performed using unpaired two‐tailed Student's t ‐test. Data are presented as mean ± SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Using 5% digitonin to permeabilised the cell membranes allowedprimary antibodies against CTCF (Millipore, Cat. No 07‐729), BRD4 (EpiCypher, Cat. No 13‐2003), H3K4me3 (Epicypher, Cat. No 13‐0041), H3K27ac (Abcam, Cat. No ab4729) and IgG‐specific (Epicypher, Cat. No 13‐0042) secondary antibodies to bind to the DNA chromatin during incubation overnight at 4 °C.

Techniques: Expressing, Western Blot, Knockdown, Control, Comparison, Co-Immunoprecipitation Assay, Immunoprecipitation, Quantitative RT-PCR, Binding Assay, Two Tailed Test

Journal: Advanced Science

Article Title: Epstein‐Barr Virus Expressed Long Non‐Coding RNA ( lncBART s) Regulate EBV Latent Genome Replication

doi: 10.1002/advs.202507286

Figure Lengend Snippet: The role of lncBART s in driving tumorigenesis by regulating MYC expression. a) Annexin V and PI staining of lncBART s and BRD4 knockdown (passage 19) and control C666‐1 cells were analyzed by flow cytometry. Experiments were repeated three times and representative images are shown. The adjacent bar chart quantifies the percentage of Annexin V positive cells. b) CCK8 assays in lncBART s knockdown (passage 18) and control C666‐1 cells for 5 consecutive days following the treatment of 2 mg/ml dox to determine cell viability (n = 3). c) Transwell invasion assay indicates the invasion abilities in lncBART s and BRD4 knockdown (passage 20) and control C666‐1 cells. Scale bar, 100 µm. d) GSEA enrichment plots, using the Hallmark gene set, demonstrate that Hallmark MYC targets v1 and MYC targets v2 are enriched in lncBART s knockdown C666‐1 compared to control cells in RNA‐seq. e) The expression levels of MYC and BCL2 in lncBART s knockdown C666‐1, YCCEL1, and NPC43‐C7‐M81 cells, along with their controls, were assessed using western blot. f) The expression levels of MYC and BCL2 in BRD4 knockdown and control C666‐1 cells were assessed using western blot. g) The expression levels of MYC and BCL2 in C666‐1 cells treated with 500 nM JQ1 or DMSO for 48 h were assessed using western blot. h) The bar graph illustrates the expression levels of RPMS1 and MYC in clusters EPI‐1 to EPI‐10. Clusters EPI‐1 to EPI‐5 are demonstrate enhancer activation, as determined through single‐cell RNA‐seq analysis. i) The Single‐cell spatial analysis with the CosMx Spatial Molecular Imager (SMI) analysis image demonstrates the expression of the RPMS1 and MYC genes within an NPC biopsy. Cell types within the tissue are identified using PanCK (epithelial cancer cells, green), CD45 (lymphocyte cells, purple) and DNA (blue) staining, as shown on the left. Clusters EPI‐1 (blue), ‐2 (light blue), ‐3 (yellow), ‐4 (brown), and ‐5 (purple) are displayed on the right. Scale bar, 100 µm. j) An enlarged view of the boxed region in H displays a single cell expressing RPMS1 (red) and MYC (blue) genes on the left, while the different cell types are shown on the right. Scale bar, 3 µm. k) The UMAP dimension reduction displays cellular maps at a single‐cell resolution, demonstrating the expression of MYC and RPMS1 . The red color represents exclusive MYC expression, the green color signifies exclusive RPMS1 expression, and the yellow color indicates co‐expression of both. Statistical analysis was performed using unpaired two‐tailed Student's t ‐test. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Using 5% digitonin to permeabilised the cell membranes allowedprimary antibodies against CTCF (Millipore, Cat. No 07‐729), BRD4 (EpiCypher, Cat. No 13‐2003), H3K4me3 (Epicypher, Cat. No 13‐0041), H3K27ac (Abcam, Cat. No ab4729) and IgG‐specific (Epicypher, Cat. No 13‐0042) secondary antibodies to bind to the DNA chromatin during incubation overnight at 4 °C.

Techniques: Expressing, Staining, Knockdown, Control, Flow Cytometry, Transwell Invasion Assay, RNA Sequencing, Western Blot, Activation Assay, Single Cell, Two Tailed Test

Journal: Advanced Science

Article Title: Epstein‐Barr Virus Expressed Long Non‐Coding RNA ( lncBART s) Regulate EBV Latent Genome Replication

doi: 10.1002/advs.202507286

Figure Lengend Snippet: Working model of the lncBART s complex in maintaining EBV episome and promoting EBV‐associated oncogenesis. This working model illustrates that lncBART s anchor a protein complex, including host factors BRD4 and CTCF along with the viral factor EBNA1, to the EBV ori P region. This process is essential in tethering EBV to host chromosomes, thereby maintaining the EBV copy number in EBV‐infected cells. Besides, lncBART s affect the expression of host proto‐oncogenes, such as MYC , thereby leading to EBV‐associated oncogenesis.

Article Snippet: Using 5% digitonin to permeabilised the cell membranes allowedprimary antibodies against CTCF (Millipore, Cat. No 07‐729), BRD4 (EpiCypher, Cat. No 13‐2003), H3K4me3 (Epicypher, Cat. No 13‐0041), H3K27ac (Abcam, Cat. No ab4729) and IgG‐specific (Epicypher, Cat. No 13‐0042) secondary antibodies to bind to the DNA chromatin during incubation overnight at 4 °C.

Techniques: Infection, Expressing

Journal: Nature Communications

Article Title: Identification of a SNAI1 enhancer RNA that drives cancer cell plasticity

doi: 10.1038/s41467-025-58032-w

Figure Lengend Snippet: a The interaction between SNAI1e and BRD4 in MDA‐MB‐231 cells was analyzed by RNA immunoprecipitation (RIP). YTHDC1 served as a negative control. RT-qPCR was performed to detect SNAI1e expression in immunoprecipitants from MDA‐MB‐231 cells. The results are expressed as mean ± SD from three independent experiments. b The interaction between SNAI1e and BRD4 in MDA-MB-231 cells was analyzed by RNA pull-down. Western blotting analysis was performed to detect FLAG expression in whole-cell lysates (Input) and immunoprecipitants (IP). LETS1 and SNAI1e-AS served as negative controls. The RNA amounts used for pull-down were evaluated by agarose gel electrophoresis. c The interaction between SNAI1e truncation mutants and BRD4 in MDA-MB-231 cells was analyzed by RNA pull-down. Western blotting analysis was performed to detect FLAG expression in whole-cell lysates (Input) and immunoprecipitants (IP). The RNA amounts used for pull-down were evaluated by agarose gel electrophoresis. d Schematic representation of full-length (FL) BRD4 and the truncation mutants tested. e The interactions between SNAI1e and BRD4 FL or the truncation mutants in MDA-MB-231 cells were analyzed by RNA pull-down. SNAI1e-AS , antisense SNAI1e ; SNAI1e-S , sense SNAI1e . Western blotting analysis was performed to detect FLAG expression in whole-cell lysates (Input) and immunoprecipitants (IP). The RNA amounts used for pull-down were evaluated by agarose gel electrophoresis. f The direct interaction between SNAI1e and the FLAG-BRD4 BD1/2 recombinant protein was analyzed by in vitro RIP. The results are expressed as mean ± SD from three independent experiments. The FLAG-tagged proteins in immunoprecipitants were evaluated by western blotting. g The direct interaction between SNAI1e and the recombinant FLAG-BRD4 BD1/2 protein was analyzed by in vitro RNA pull-down. Western blotting analysis was performed to detect FLAG expression in whole-cell lysates (Input) and immunoprecipitants (IP). The RNA amounts used for pull-down were evaluated by agarose gel electrophoresis. Significance was calculated by using one-way ANOVA followed by Dunnett’s ( a ) and Tukey’s ( f ) multiple comparisons test. Data are representative of at least three ( b , c , e , g ) independent experiments with similar results. Co.vec empty control vector.

Article Snippet: For in vitro RIP, 10 pmol of in vitro-transcribed SNAI1e was incubated with 2 μg recombinant FLAG-SMURF2 protein (Sigma‒Aldrich; SRP0228) or FLAG-BRD4 BD1/2 recombinant protein (R&D systems; SP-600) for 16 h at 4 °C as described previously .

Techniques: RNA Immunoprecipitation, Negative Control, Quantitative RT-PCR, Expressing, Western Blot, Agarose Gel Electrophoresis, Recombinant, In Vitro, Control, Plasmid Preparation

Journal: Theranostics

Article Title: Divergent splicing factor SRSF1 signaling promotes inflammation post-CME: the SRSF1/ENPP3 axis acts via inhibition of BRD4 O-GlcNAcylation to enhance NF-κB activation and accelerate heart failure

doi: 10.7150/thno.115402

Figure Lengend Snippet: O-GlcNAcylation of BRD4 inhibited NF-κB p65-mediated transcription of pro-inflammatory cytokines. (A)&(B) The expression of BRD4 in OGD-exposed cardiomyocytes was detected by RT-qPCR and Western blotting. H9C2 and AC-16 cells were transfected with shBRD4, and then subjected to OGD. (C)&(D) RT-qPCR and Western blotting analysis of BRD4 mRNA and protein levels. (E)&(F) The mRNA levels and concentrations of TNF-α, IL-1β, and IL-6 were determined by RT-qPCR and ELISA. (G) The binding of NF-κB p65 to TNF-α, IL-1β, and IL-6 promoters was confirmed by dual-luciferase reporter assay. (H)&(I) Co-IP assay verified the exogenous and endogenous interplay between OGT and BRD4 proteins. (J) O-GlcNAcylation of BRD4 protein in OGD-stimulated cardiomyocytes was evaluated. (K) YinOYang database predicated the potential O-GlcNAc sites on BRD4. OGD-challenged H9C2 and AC-16 cells were transfected with BRD4 WT plasmid or BRD4 plasmids with mutant O-GlcNAc sites (BRD4-S484R, BRD4-S784R, and BRD4-T1212R). (L) O-GlcNAcylation of BRD4 protein in H9C2 and AC-16 cells was detected. (M) Concentrations of TNF-α, IL-1β, and IL-6 were detected by ELISA. (N) The interaction between NF-κB p65 and TNF-α, IL-1β, and IL-6 promoters was validated by dual-luciferase reporter assay. n=3 for A-N. Student's t test (for A, B) and one-way ANOVA (for C-G, M, N) were performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The sections received overnight incubation with primary antibodies SRSF1 (12929-2-AP, 1:50, Proteintech, Wuhan, China), ENPP3 (A05615, 1:100, Boster, CA, USA), or BRD4 (M00123, 1:50, Boster) at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Binding Assay, Luciferase, Reporter Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Mutagenesis

Journal: Theranostics

Article Title: Divergent splicing factor SRSF1 signaling promotes inflammation post-CME: the SRSF1/ENPP3 axis acts via inhibition of BRD4 O-GlcNAcylation to enhance NF-κB activation and accelerate heart failure

doi: 10.7150/thno.115402

Figure Lengend Snippet: ENPP3 contributed to inflammation by inhibiting O-GlcNAcylation of BRD4. H9C2 and AC-16 cells were transfected with shENPP3, followed by exposure to OGD. (A) ENPP3 and BRD4 protein levels were measured by Western blotting. (B) The O-GlcNAc level of BRD4 protein was assessed. (C) The production of TNF-α, IL-1β, and IL-6 was determined by ELISA. (D) Dual-luciferase reporter assay evaluated the binding of NF-κB p65 to TNF-α, IL-1β, and IL-6 promoters. n=3 for A-D. One-way ANOVA was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The sections received overnight incubation with primary antibodies SRSF1 (12929-2-AP, 1:50, Proteintech, Wuhan, China), ENPP3 (A05615, 1:100, Boster, CA, USA), or BRD4 (M00123, 1:50, Boster) at 4 °C.

Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Binding Assay

Journal: Theranostics

Article Title: Divergent splicing factor SRSF1 signaling promotes inflammation post-CME: the SRSF1/ENPP3 axis acts via inhibition of BRD4 O-GlcNAcylation to enhance NF-κB activation and accelerate heart failure

doi: 10.7150/thno.115402

Figure Lengend Snippet: SRSF1/ENPP3 axis suppressed BRD4 O-GlcNAcylation to promote inflammation in CME. The OGD-stimulated cardiomyocytes were transfected with shSRSF1, ENPP3 overexpression plasmid, or a combination of them. (A) ENPP3 mRNA and lncRNA ENPP3 expression levels were detected by RT-qPCR. (B) The protein abundance of ENPP3 and BRD4 was assessed by Western blotting. (C) The O-GlcNAc level of BRD4 was determined. (D) ELISA was carried out to measure TNF-α, IL-1β, and IL-6 concentrations. n=3 for A-D. One-way ANOVA was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The sections received overnight incubation with primary antibodies SRSF1 (12929-2-AP, 1:50, Proteintech, Wuhan, China), ENPP3 (A05615, 1:100, Boster, CA, USA), or BRD4 (M00123, 1:50, Boster) at 4 °C.

Techniques: Transfection, Over Expression, Plasmid Preparation, Expressing, Quantitative RT-PCR, Quantitative Proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Divergent splicing factor SRSF1 signaling promotes inflammation post-CME: the SRSF1/ENPP3 axis acts via inhibition of BRD4 O-GlcNAcylation to enhance NF-κB activation and accelerate heart failure

doi: 10.7150/thno.115402

Figure Lengend Snippet: Myocardium-specific SRSF1 knockout alleviated CME-induced inflammation via inactivation of the ENPP3/BRD4/NF-κB pathway. SRSF1 flox/flox and SRSF1-KO rats were injected with microspheres into the left ventricle to induce CME. (A) LVEF, LVFS, LVEDd, and CO were detected to evaluate cardiac function. (B) The serum cTnl level in different groups was measured by ELISA. (C) Pathological alterations in myocardial tissues were observed by HE staining (scale bar = 100 μm). (D) Myocardial infarct size was measured by HBFP staining (scale bar = 100 μm). (E) SRSF1, ENPP3, and BRD4 expression in myocardial tissues was evaluated by immunohistochemical staining (scale bar = 100 μm). (F) The protein abundance of SRSF1, ENPP3, BRD4, p65, and O-GlcNAcylation of BRD4 was detected by Western blotting or Co-IP, respectively. (G) ELISA was carried out to measure TNF-α, IL-1β, and IL-6 concentrations. n=6 for A-G. ANOVA for repeated measurement (for A, B), and one-way ANOVA (for F, G) was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The sections received overnight incubation with primary antibodies SRSF1 (12929-2-AP, 1:50, Proteintech, Wuhan, China), ENPP3 (A05615, 1:100, Boster, CA, USA), or BRD4 (M00123, 1:50, Boster) at 4 °C.

Techniques: Knock-Out, Injection, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Immunohistochemical staining, Quantitative Proteomics, Western Blot, Co-Immunoprecipitation Assay