Journal: Frontiers in Pharmacology

Article Title: Epigenetic Reader Bromodomain Containing Protein 2 Facilitates Pathological Cardiac Hypertrophy via Regulating the Expression of Citrate Cycle Genes

doi: 10.3389/fphar.2022.887991

Figure Lengend Snippet: BRD2 expression was elevated in ISO-induced cardiac hypertrophy. Hypertrophic changes of left ventricle in ISO-treated rats were observed by (A) gross morphologic examination and (B) echocardiography. (C) The mRNA and (D) protein expression of BRD2, ANF and BNP in ISO-treated rats. (E) The mRNA and (F) protein expression of BRD2, ANF and BNP after treatment of 10 μM ISO for indicated times in cardiomyocytes. The data were presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control, **** p < 0.0001 vs. control. n = 4.

Article Snippet: Antibodies against BRD2 (bromodomain containing 2, Cat# A302-583A-M) were from Bethyl; anti-α-tubulin (Cat# 66031-1-IG) and GAPDH (Cat# 60004-1-IG) were obtained from Proteintech; anti-ANF (atrial natriuretic factor, Cat# PAA225Ra02) was bought from Cloud-Clone Corp. Normal HRP-conjugated secondary antibodies were obtained from cell Signaling Technology.

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Epigenetic Reader Bromodomain Containing Protein 2 Facilitates Pathological Cardiac Hypertrophy via Regulating the Expression of Citrate Cycle Genes

doi: 10.3389/fphar.2022.887991

Figure Lengend Snippet: The effects of BRD2 in ISO-induced cardiomyocyte hypertrophy. (A) The mRNA level and (B) protein expression of BRD2, ANF and BNP, and (C) cell surface area measured by rhodamine-phalloidin staining, in cardiomyocytes transfected with BRD2 plasmid or blank vector for 48 h. Scale bar: 20 μm. (D) The mRNA level and (E) protein expression of BRD2, ANF and BNP, and (F) cell surface area measured by rhodamine-phalloidin staining, in cardiomyocytes transfected with siRNA targeting BRD2 (siBRD2) or negative control (NC) for 48 h, and further incubated with 10 μM ISO or DMSO for 24 h. The data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ISO. n = 4.

Article Snippet: Antibodies against BRD2 (bromodomain containing 2, Cat# A302-583A-M) were from Bethyl; anti-α-tubulin (Cat# 66031-1-IG) and GAPDH (Cat# 60004-1-IG) were obtained from Proteintech; anti-ANF (atrial natriuretic factor, Cat# PAA225Ra02) was bought from Cloud-Clone Corp. Normal HRP-conjugated secondary antibodies were obtained from cell Signaling Technology.

Techniques: Expressing, Staining, Transfection, Plasmid Preparation, Negative Control, Incubation

Journal: Frontiers in Pharmacology

Article Title: Epigenetic Reader Bromodomain Containing Protein 2 Facilitates Pathological Cardiac Hypertrophy via Regulating the Expression of Citrate Cycle Genes

doi: 10.3389/fphar.2022.887991

Figure Lengend Snippet: BRD2 knockdown protected SD rats from ISO-induced cardiac hypertrophy. SD rats received intramyocardial injections of adeno-associated virus encoding siBRD2 or si-NC, followed by subcutaneous injection of ISO (1.2 mg/kg/d) or an equal volume of normal saline (NS) 2 weeks after operation for 7 days. Hypertrophic changes of left ventricle were observed by (A) gross morphologic examination and (B) H&E staining, (C) PSR staining and (D) echocardiography. Scar bar: 50 μm. Post-mortem measurements of (E) heart weight/body weight (HW/BW) and (F) heart weight/tibia length (HW/TL) values. Echocardiography measurements of (G) ejection fraction (EF, %) and (H) fraction shortening (FS, %) values. (I) The protein expression of BRD2 and ANF measured by Western blot. The data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 vs . control. **** p < 0.0001 vs. control. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ISO, ### p < 0.001 vs. ISO, #### p < 0.0001 vs. vs. ISO. n = 7.

Article Snippet: Antibodies against BRD2 (bromodomain containing 2, Cat# A302-583A-M) were from Bethyl; anti-α-tubulin (Cat# 66031-1-IG) and GAPDH (Cat# 60004-1-IG) were obtained from Proteintech; anti-ANF (atrial natriuretic factor, Cat# PAA225Ra02) was bought from Cloud-Clone Corp. Normal HRP-conjugated secondary antibodies were obtained from cell Signaling Technology.

Techniques: Injection, Staining, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Epigenetic Reader Bromodomain Containing Protein 2 Facilitates Pathological Cardiac Hypertrophy via Regulating the Expression of Citrate Cycle Genes

doi: 10.3389/fphar.2022.887991

Figure Lengend Snippet: RNA sequencing analysis of transcriptome changes in NRCMs with BRD2 overexpression or silencing. (A) Venn diagram showing genes differentially expressed between “BRD2 vs. CON” and “siBRD2+ISO vs. ISO” groups (|log 2 FC| ≥ 0.5, Q value ≤ 0.05). (B) Heatmap showing differentially expressed genes between “BRD2-vs-CON” and “siBRD2+ISO” vs. “ISO” groups (|log 2 FC| ≥ 0.5, Q value ≤ 0.05). (C) KEGG pathway analysis of 314 genes regulated by BRD2 (|log 2 FC| ≥ 0.5, Q value ≤ 0.05). (D) Heatmap showing the differentially expressed metabolic genes under regulation by BRD2 according to the RNA-seq data (|log 2 FC| ≥ 0.5, Q value ≤ 0.05). (E) GSEA analysis of differentially expressed genes between “BRD2″ vs. “CON” group.

Article Snippet: Antibodies against BRD2 (bromodomain containing 2, Cat# A302-583A-M) were from Bethyl; anti-α-tubulin (Cat# 66031-1-IG) and GAPDH (Cat# 60004-1-IG) were obtained from Proteintech; anti-ANF (atrial natriuretic factor, Cat# PAA225Ra02) was bought from Cloud-Clone Corp. Normal HRP-conjugated secondary antibodies were obtained from cell Signaling Technology.

Techniques: RNA Sequencing Assay, Over Expression

Journal: Frontiers in Pharmacology

Article Title: Epigenetic Reader Bromodomain Containing Protein 2 Facilitates Pathological Cardiac Hypertrophy via Regulating the Expression of Citrate Cycle Genes

doi: 10.3389/fphar.2022.887991

Figure Lengend Snippet: BRD2 modulated expression of TCA cycle genes and energy production during ISO-induced cardiac hypertrophy. qPCR measurement of TCA cycle genes in (A) NRCMs with BRD2 overexpression or knockdown, and (B) NRCMs with or without BRD2 knockdown or ISO treatment. (C) Schematic diagram showing TCA genes changes following ISO treatment in NRCMs. (D) Oxygen consumption rates (OCR) measured by XF Cell Mito Stress Test Kit. (E) ATP production in cardiomyocytes with BRD2 overexpression or knockdown. The data are presented as mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control, **** p < 0.0001 vs. control. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. ISO, #### p < 0.0001 vs. ISO. n = 5.

Article Snippet: Antibodies against BRD2 (bromodomain containing 2, Cat# A302-583A-M) were from Bethyl; anti-α-tubulin (Cat# 66031-1-IG) and GAPDH (Cat# 60004-1-IG) were obtained from Proteintech; anti-ANF (atrial natriuretic factor, Cat# PAA225Ra02) was bought from Cloud-Clone Corp. Normal HRP-conjugated secondary antibodies were obtained from cell Signaling Technology.

Techniques: Expressing, Over Expression

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: BET family genes are differentially induced by CoV infection. ( A ) H1299, DF1, IPEC-J2, and Vero cells were infected with IBV, PEDV, HCoV-OC43, or HCoV-229E (MOI ~2) or mock-treated with UV-inactivated viruses. Total RNA was extracted at the indicated time points, and RT-qPCR was performed to quantify viral genomic RNA (CoV gRNA) and BET family mRNAs (BRD2, BRD3, BRD4, and BRDT). Data were normalized to GAPDH mRNA (0 hpi) using the ΔΔCt method (ns, non-significant; *, P <0.05; **, P < 0.01; ***, P < 0.001). N.D., non-determined. ( B ) Cells were infected or mock-treated as in panel A , harvested at the indicated times, and analyzed by Western blot with the specified antibodies. β-actin served as a loading control. Protein ladder sizes (kDa) are shown.

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Control

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: Interaction of the C-terminal region of BRD2 protein with the C-terminal region of CoV E protein. ( A ) 293T cells were transfected with pXJ40-FLAG, pXJ40-IBV E-FLAG, pXJ40-IBV E-T16A-FLAG, pXJ40-IBV E-A26F-FLAG, or pXJ40-BRD2. After 24 h, lysates were immunoprecipitated (IP) with anti-FLAG beads and analyzed by Western blot (anti-FLAG/anti-BRD2). Protein ladder sizes (kDa) are shown. Sizes of protein ladders in kDa were indicated on the left. ( B ) Diagram showing the putative functional domains in the full-length and three truncated BRD2 constructs, BRD2-N1, BRD2-C1, and BRD2-C2. ( C ) 293T cells were transfected with pXJ40-FLAG, pXJ40-IBV E-FLAG, or myc-tagged BRD2 constructs (full-length, N1, C1, C2). Lysates were immunoprecipitated with anti-FLAG and probed with anti-FLAG/anti-myc. Sizes of protein ladders in kDa were indicated on the left. ( D ) Diagram showing the putative functional domains in the full-length and two truncated IBV E constructs, IBV E-N, IBV E-C. ( E ) 2293T cells expressing myc-BRD2-C1 with GFP-tagged IBV E (full-length, N-, or C-terminus) were immunoprecipitated with anti-myc beads and blotted with anti-myc/anti-GFP. Sizes of protein ladders in kDa were indicated on the left.

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: Transfection, Immunoprecipitation, Western Blot, Functional Assay, Construct, Expressing

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: Re-localization of BRD2 from the nucleus to the cytoplasm in CoV-infected cells. ( A ) Cells infected with IBV (MOI~2) or mock-treated (UV-inactivated virus) were fixed at 0, 8, and 12 hpi, permeabilized, and immunostained with rabbit anti-BRD2 and mouse anti-IBV N/dsRNA. IBV N/dsRNA (Alexa Fluor 488, green), BRD2 (Alexa Fluor 594, red), and nuclei (DAPI/Hoechst 33,342, blue) were visualized. ( B ) Vero cells were either infected with IBV at an MOI~2 or mock-treated with UV-inactivated IBV and harvested at the indicated times post-infection. Nuclear and cytosolic fractions were prepared and subjected to western blotting with the indicated antibodies. Sizes of protein ladders in kDa were indicated on the left. ( C ) The grayscale values of protein bands shown in panel B were measured using Image J. The relative protein expression level of BRD2 in the nuclear fraction of the mock group was set as 100%, and the levels in other groups were calculated as percentages relative to this value.

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: Infection, Virus, Western Blot, Expressing

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: The association between the host protein BRD2 and IBV virions. ( A ) IBV-infected Vero cell supernatants were purified by sucrose gradient 20%. The sucrose pellet was analyzed by Western blot with the indicated antibodies. β-actin served as a loading control; protein ladder sizes (kDa) are shown. ( B ) IBV-infected Vero cell supernatants were purified by sucrose gradient (20%–60%). Fractions (12 total) were analyzed by Western blot with the indicated antibodies. Nine of twelve fractions (lanes 2–10) were 10× concentrated and analyzed by Western blot (antibodies indicated). β-actin was the loading control; protein ladder sizes in kDa are marked. ( C ) IBV-infected Vero cell supernatants were purified by sucrose gradient 20%. The sucrose pellet was immunoprecipitated with anti-IBV S/N/M/E and probed with anti-IBV S/N/M/E or anti-BRD2. Sizes of protein ladders in kDa were indicated on the left.

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: Infection, Purification, Western Blot, Control, Immunoprecipitation

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: BRD2 protein regulates CoV replication and apoptosis. ( A ) Cell viability of H1299-shNC/BRD2 and Vero-shNC/BRD2 cells was assessed by CCK-8 assay (A450). Significance levels were presented by the p -value (ns, non-significant; *, P <0.05; **, P < 0.01; ***, P < 0.001). N.D., non-determined. ( B ) IBV titers (log TCID50/ml) in lysates/supernatants of infected H1299-shNC/BRD2 and Vero-shNC/BRD2 cells at the indicated time points. Significance as described for panel A . ( C ) Western blot analysis of IBV-infected (MOI ~2) or mock-treated H1299-shNC/BRD2 and Vero-shNC/BRD2 cells using the indicated antibodies. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left. ( D ) IBV-infected or mock-infected H1299 cells pre-transfected with siEGFP/siBRD2 were analyzed by Western blot. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left. ( E ) Western blot of IBV-infected or mock-infected H1299 cells transfected with pXJ40-FLAG/pXJ40-FLAG-BRD2. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left.

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: CCK-8 Assay, Infection, Western Blot, Control, Transfection

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: BRD2 promotes the induction of pro-inflammatory response in CoV-infected cells. ( A ) IBV-infected (MOI ~2) or mock-treated HeLa, DF1, and Vero cells were analyzed by RT-qPCR for viral gRNA and cytokine mRNAs (IL-6, IL-8, TNF-α), normalized to 0 hpi GAPDH. Significance levels were presented by the P -value (ns, non-significant; *, P <0.05; **, P < 0.01; ***, P < 0.001). N.D., non-determined. ( B ) Western blot analysis of IBV-infected or mock-treated HeLa, DF1, and Vero cells at the indicated time points. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left. ( C ) RT-qPCR analysis of IBV RNAs (gRNA, sgRNA), cytokines (IL-6, IL-8, TNF-α), and BET family mRNAs (BRD2/3/4, BRDT) in H1299/Vero-shNC/BRD2 cells, normalized to GAPDH in shNC at 0 hpi. Significance as described for panel A . ( D ) IBV-infected or mock-infected H1299 cells pre-transfected with siEGFP/siBRD2 were analyzed by RT-qPCR for viral gRNA, cytokines, and BET mRNAs, normalized to GAPDH in siEGFP mock at 16 hpi. Significance as described for panel A .

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Control, Transfection

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: BRD2 protein promotes the activation of the NF-κB signaling pathway and the expression of pro-inflammatory factors. ( A ) H1299 cells transfected with pXJ40-FLAG or pXJ40-FLAG-BRD2 were infected with IBV or mock-treated, then analyzed by Western blot. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left. The FLAG, IBV N, and β-actin loading control shown here are from the same experimental batch as the samples in and are reused to ensure a consistent baseline for cross-figure comparison. ( B ) As in panel A , but infected with HCoV-OC43. Beta-actin was included as the loading control. Sizes of protein ladders in kDa were indicated on the left. ( C ) H1299 and Vero cells transfected as described for panel A were infected with IBV, PEDV, HCoV-OC43, or HCoV-229E. RT-qPCR measured viral gRNA, cytokine mRNAs (IL-6/8, TNF-α), and BET family transcripts (BRD2/3/4, BRDT), normalized to FLAG-transfected mock 16 hpi GAPDH. Significance levels were presented by the P -value (ns, non-significant; *, P <0.05; **, P < 0.01; ***, P < 0.001). N.D., non-determined.

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: Activation Assay, Expressing, Transfection, Infection, Western Blot, Control, Comparison, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Interaction of coronavirus E protein with BRD2 plays important regulatory roles in viral replication and induction of pro-inflammatory response

doi: 10.1128/jvi.02201-25

Figure Lengend Snippet: Diagram illustrating the current working model. Working model of BRD2-IκBα-NF-κB axis activation of pro-inflammatory cytokines/chemokines and BRD2-E protein interaction during CoV infection. Arrowheads indicate activation; blunt ends indicate suppression. Dashed lines represent incompletely characterized processes.

Article Snippet: Antibodies against BRD2 (#5848), NF-κB p65 (#8242), Phospho-NF-κB p65 (#3033), IκBα (#4814), Phospho-IκBα (#5209), IL-6 (#12912), FLAG-Tag (#8146), GFP-Tag (#2956), myc-Tag (#2276), H3 (#4499), α-Tubulin (#3873), and β-actin (#4967) were from Cell Signaling Technology.

Techniques: Activation Assay, Infection

Journal: Cancer Communications

Article Title: BRD2 induces drug resistance through activation of the RasGRP1/Ras/ERK signaling pathway in adult T‐cell lymphoblastic lymphoma

doi: 10.1002/cac2.12039

Figure Lengend Snippet: The association between BRD2 mRNA level and clinicopathological characteristics in 85 adult T‐LBL patients

Article Snippet: Then, the slides were incubated with primary antibodies against BRD2 (1:100; sc‐393720; Santa Cruz Biotechnology) and c‐Myc (1:200; sc‐40; Santa Cruz Biotechnology) overnight at 4°C, and subsequently incubated with the secondary antibody mouse IgGκ binding protein‐HRP (1:100; sc‐393720; Santa Cruz Biotechnology) for 30 min at 37°C.

Techniques: Concentration Assay, Mutagenesis

Journal: Cancer Communications

Article Title: BRD2 induces drug resistance through activation of the RasGRP1/Ras/ERK signaling pathway in adult T‐cell lymphoblastic lymphoma

doi: 10.1002/cac2.12039

Figure Lengend Snippet: BRD2 is upregulated in chemotherapy‐resistant T‐LBL tissues and associated with poor prognosis. A. Heatmap of differentially expressed coding mRNAs was obtained by hierarchical clustering analysis with the average linkage method. DS indicates chemotherapy‐sensitive T‐LBL tissues; DR indicates chemotherapy‐resistant T‐LBL tissues. B. The relative mRNA expression level of BRD2 was detected by semi‐quantitative real‐time PCR in 85 fresh‐frozen T‐LBL tissue samples. *, P < 0.05. C. The protein expression of BRD2 was detected by immunohistochemistry in 85 fresh‐frozen T‐LBL samples. D and E. ROC curve analysis was used to determine the optimal cut‐off value of BRD2 mRNA/protein expression level (left panel). High mRNA/protein expression level of BRD2 was associated with poor prognosis of T‐LBL patients (right panel). Kaplan‐Meier analysis was used to estimate PFS curves ( P < 0.001, log‐rank test). Abbreviations: T‐LBL, T‐cell lymphoblastic lymphoma; ROC, receiver operating characteristic; HR, hazard ratio; CI, confidence interval; PFS, progression‐free survival

Article Snippet: Then, the slides were incubated with primary antibodies against BRD2 (1:100; sc‐393720; Santa Cruz Biotechnology) and c‐Myc (1:200; sc‐40; Santa Cruz Biotechnology) overnight at 4°C, and subsequently incubated with the secondary antibody mouse IgGκ binding protein‐HRP (1:100; sc‐393720; Santa Cruz Biotechnology) for 30 min at 37°C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry

Journal: Cancer Communications

Article Title: BRD2 induces drug resistance through activation of the RasGRP1/Ras/ERK signaling pathway in adult T‐cell lymphoblastic lymphoma

doi: 10.1002/cac2.12039

Figure Lengend Snippet: BRD2 induces drug resistance in T‐LBL cells in vitro . A. Relative BRD2 mRNA expression levels in Jurkat and SUP‐T1 cells stably transfected with empty vectors and BRD2‐overexpressing plasmids. B. Cell viability detected by MTT assay in control and BRD2‐overexpressing Jurkat and SUP‐T1 cells treated with Dox (100 ng/mL, 12 h). C. Dox‐induced apoptosis was detected by Annexin V/PI staining and flow cytometry assay in control and BRD2‐overexpressing Jurkat and SUP‐T1 cells. D. The protein expression levels of Bax and Bcl‐2 were examined by Western blotting in control and BRD2‐overexpressing Jurkat and SUP‐T1 cells treated with Dox (100 ng/mL, 12 h). The protein expression of GAPDH was used as a loading control. Results are presented as mean ± standard deviation (SD) of three independent experiments; *, P < 0.05. Abbreviations: T‐LBL, T‐cell lymphoblastic lymphoma; Dox, doxorubicin; SD, standard deviation

Article Snippet: Then, the slides were incubated with primary antibodies against BRD2 (1:100; sc‐393720; Santa Cruz Biotechnology) and c‐Myc (1:200; sc‐40; Santa Cruz Biotechnology) overnight at 4°C, and subsequently incubated with the secondary antibody mouse IgGκ binding protein‐HRP (1:100; sc‐393720; Santa Cruz Biotechnology) for 30 min at 37°C.

Techniques: In Vitro, Expressing, Stable Transfection, Transfection, MTT Assay, Control, Staining, Flow Cytometry, Western Blot, Standard Deviation

Journal: Cancer Communications

Article Title: BRD2 induces drug resistance through activation of the RasGRP1/Ras/ERK signaling pathway in adult T‐cell lymphoblastic lymphoma

doi: 10.1002/cac2.12039

Figure Lengend Snippet: BRD2 induces drug resistance in T‐LBL xenograft in mice. The xenografts were formed by SUP‐T1‐GFP or SUP‐T1‐BRD2‐GFP cells and treated with Dox (1 mg/kg/time, 3 times/week). A. The fluorescent signal of xenograft was detected by in vivo fluorescence imaging system. B. Tumor size of xenografts was measured every 4 days for 52 days. C . Xenografts resected at day 52 after injections of cells. D. The protein expression levels of Bax and Bcl‐2 in xenografts treated with Dox were examined by Western blotting. The protein expression of GAPDH was used as a loading control. *, P < 0.05. Abbreviations: T‐LBL, T‐cell lymphoblastic lymphoma; Dox, doxorubicin

Article Snippet: Then, the slides were incubated with primary antibodies against BRD2 (1:100; sc‐393720; Santa Cruz Biotechnology) and c‐Myc (1:200; sc‐40; Santa Cruz Biotechnology) overnight at 4°C, and subsequently incubated with the secondary antibody mouse IgGκ binding protein‐HRP (1:100; sc‐393720; Santa Cruz Biotechnology) for 30 min at 37°C.

Techniques: In Vivo, Fluorescence, Imaging, Expressing, Western Blot, Control

Journal: Cancer Communications

Article Title: BRD2 induces drug resistance through activation of the RasGRP1/Ras/ERK signaling pathway in adult T‐cell lymphoblastic lymphoma

doi: 10.1002/cac2.12039

Figure Lengend Snippet: BRD2 induces drug resistance of T‐LBL by activating the MEK/ERK/c‐Myc signaling pathway. A. Protein levels of p‐ERK, ERK, p‐MEK, MEK, and c‐Myc were detected by Western blotting in Jurkat and SUP‐T1 cells that stably transfected with empty vector and BRD2‐expressing plasmids. The protein expression of β‐actin was used as loading control. B. Representative images of c‐Myc and BRD2 IHC staining in adult T‐LBL tissues. IHC staining was performed to detect c‐Myc‐ and BRD2‐positive cells in 85 adult T‐LBL tissues. C. MTT assay was used to detect the viability of Jurkat and SUP‐T1 cells that stably transfected with empty vector and BRD2‐expressing plasmids and underwent Dox (100 ng/mL, 12 h) and/or PD098059 (10 µmol/L, 12 h) treatment. Results are presented as mean ± SD of three independent experiments; *, comparison between Control + Dox group and BRD2 + Dox group, P < 0.05; #, comparison between BRD2 + Dox group and BRD2 + Dox + PD098059 group, P < 0.05. Abbreviations: T‐LBL, T‐cell lymphoblastic lymphoma; IHC; immunohistochemistry; Dox, doxorubicin; SD, standard deviation

Article Snippet: Then, the slides were incubated with primary antibodies against BRD2 (1:100; sc‐393720; Santa Cruz Biotechnology) and c‐Myc (1:200; sc‐40; Santa Cruz Biotechnology) overnight at 4°C, and subsequently incubated with the secondary antibody mouse IgGκ binding protein‐HRP (1:100; sc‐393720; Santa Cruz Biotechnology) for 30 min at 37°C.

Techniques: Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Control, Immunohistochemistry, MTT Assay, Comparison, Standard Deviation

Journal: Cancer Communications

Article Title: BRD2 induces drug resistance through activation of the RasGRP1/Ras/ERK signaling pathway in adult T‐cell lymphoblastic lymphoma

doi: 10.1002/cac2.12039

Figure Lengend Snippet: BRD2 directly upregulates RasGRP1 expression in T‐LBL. A. RasGTP protein is enriched in BRD2‐overexpressing T‐LBL cells. The enrichment of RasGTP and total Ras was detected by Ras pull‐down assay. B. Western blotting assay reveals that the protein level of RasGRP1 is increased in BRD2‐overexpressing T‐LBL cells. The protein expression of GAPDH was used as a loading control. C. Semi‐quantitative real‐time PCR reveals that the relative mRNA level of RasGRP1 is increased in BRD2‐overexpressing T‐LBL cells. Results are presented as mean ± SD of three independent experiments; *, P < 0.05. D. The putative E2F‐1/BRD2 binding site in RasGRP1 promoter sequence was detected using the online PROMO algorithm tool (left panel). ChIP assay was used to confirm the binding of BRD2 with RasGRP1 promoter (right panel). Input DNA was used as a positive control. Abbreviations: T‐LBL, T‐cell lymphoblastic lymphoma; CHIP; chromatin immunoprecipitation; SD, standard deviation

Article Snippet: Then, the slides were incubated with primary antibodies against BRD2 (1:100; sc‐393720; Santa Cruz Biotechnology) and c‐Myc (1:200; sc‐40; Santa Cruz Biotechnology) overnight at 4°C, and subsequently incubated with the secondary antibody mouse IgGκ binding protein‐HRP (1:100; sc‐393720; Santa Cruz Biotechnology) for 30 min at 37°C.

Techniques: Expressing, Pull Down Assay, Western Blot, Control, Real-time Polymerase Chain Reaction, Binding Assay, Sequencing, Positive Control, Chromatin Immunoprecipitation, Standard Deviation

Journal: Cancer Communications

Article Title: BRD2 induces drug resistance through activation of the RasGRP1/Ras/ERK signaling pathway in adult T‐cell lymphoblastic lymphoma

doi: 10.1002/cac2.12039

Figure Lengend Snippet: OTX015 suppresses the drug resistance induced by BRD2 in T‐LBL cells. A. The cell viability was measured by MTT assay in control cells (Jurkat and SUP‐T1), Dox‐treated cells (Jurkat+Dox and SUP‐T1+Dox), BRD2‐overexpressing cells treated with Dox (Jurkat‐BRD2+Dox and SUP‐T1‐BRD2+Dox), and BRD2‐overexpressing cells treated with Dox and OTX015 (Jurkat‐BRD2+Dox+OTX015 and SUP‐T1‐BRD2+Dox+OTX015). Results are presented as mean ± SD of three independent experiments; *, comparison between control group and BRD2‐overexpressing group, P < 0.05; #, comparison between BRD2‐overexpressing group and BRD2‐overexpressing + OTX015 group, P < 0.05. B. Dox‐induced apoptosis was detected by Annexin V/PI staining and flow cytometry assays in control BRD2‐overexpressing Jurkat and SUP‐T1 cells (Control), BRD2‐overexpressing Jurkat and SUP‐T1 cells treated with Dox (+Dox), and BRD2‐overexpressing Jurkat and SUP‐T1 cells treated with Dox and OTX015 (+Dox+OTX015). C. The protein expression levels of p‐ERK, ERK, p‐MEK, MEK, RasGRP1, and c‐Myc were detected by Western blotting in BRD2‐overexpressing Jurkat and SUP‐T1 cells treated with Dox alone or both Dox and OTX015. The protein expression of β‐actin was used as loading control. D. The relative mRNA levels of RasGRP1 were measured by semi‐quantitative real‐time PCR assay in BRD2‐overexpressing Jurkat and SUP‐T1 cells treated with Dox alone or both Dox and OTX015. Results are presented as mean ± SD of three independent experiments; *, P < 0.05. Abbreviations: T‐LBL, T‐cell lymphoblastic lymphoma; Dox, doxorubicin; SD, standard deviation

Article Snippet: Then, the slides were incubated with primary antibodies against BRD2 (1:100; sc‐393720; Santa Cruz Biotechnology) and c‐Myc (1:200; sc‐40; Santa Cruz Biotechnology) overnight at 4°C, and subsequently incubated with the secondary antibody mouse IgGκ binding protein‐HRP (1:100; sc‐393720; Santa Cruz Biotechnology) for 30 min at 37°C.

Techniques: MTT Assay, Control, Comparison, Staining, Flow Cytometry, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Cancer Communications

Article Title: BRD2 induces drug resistance through activation of the RasGRP1/Ras/ERK signaling pathway in adult T‐cell lymphoblastic lymphoma

doi: 10.1002/cac2.12039

Figure Lengend Snippet: The comparison of simultaneous and sequential treatment of Dox combined with OTX015 using a PDX model of T‐LBL. A. The flowchart of simultaneous and sequential treatment of Dox combined with OTX015 on PDX model. B. PDXs subjected to sequential treatment were smaller at day 36 than those subjected to simultaneous treatment. C. Tumor size of xenograft was measured every 4 days for 36 days. The mean tumor volume in the drug combination group (Dox + OTX015) was significantly smaller than that of the Dox group. D. The mean tumor volume of the simultaneous treatment group was significantly larger than that of the sequential treatment group (left panel). Right: The protein expression levels of BRD2 and c‐Myc were detected by Western blotting in xenografts. *, P < 0.05. Abbreviations: T‐LBL, T‐cell lymphoblastic lymphoma; Dox, doxorubicin; PTX, patient‐derived xenograft

Article Snippet: Then, the slides were incubated with primary antibodies against BRD2 (1:100; sc‐393720; Santa Cruz Biotechnology) and c‐Myc (1:200; sc‐40; Santa Cruz Biotechnology) overnight at 4°C, and subsequently incubated with the secondary antibody mouse IgGκ binding protein‐HRP (1:100; sc‐393720; Santa Cruz Biotechnology) for 30 min at 37°C.

Techniques: Comparison, Expressing, Western Blot, Derivative Assay