pcdna5 flag brd4 wt  (Addgene inc)

Journal: Acta Pharmaceutica Sinica. B

Article Title: BRD4 regulates m 6 A of ESPL1 mRNA via interaction with ALKBH5 to modulate breast cancer progression

doi: 10.1016/j.apsb.2024.12.037

Figure Lengend Snippet: ALKBH5 is involved in BRD4-regulated m 6 A and malignancy of BC cells. (A) The colonization capability of MDA-MB-231 and BT-549 cells treated with increasing concentrations of JQ1 were checked; (B) MDA-MB-231 cells were pre-treated with or without 0.2, 0.5, and 1 μmol/L JQ1 for 12 h, and further treated with increase concentration of Dox or CDDP for 48 h; (C) The m 6 A/A ratio of mRNA in MDA-MB-231 and BT-549 cells treated with 1 μmol/L JQ1 for 24 or 48 h was checked by LC–MS/MS; (D) The m 6 A/A ratio of mRNA in MDA-MB-231 cells transfected with si-NC or si-BRD2/3/4 for 24 h was checked by LC–MS/MS; (E) The m 6 A/A ratio of mRNA in cells transfected with vector control or BRD4 constructs for 24 h was checked by LC–MS/MS; (F) The m 6 A/A ratio of mRNA in MDA-MB-231 cells transfected with si-NC or si-BRD2/3/4 or treated with 1 μmol/L JQ1for 24 h was checked by dot-blot assay; (G) Protein expression in cells treated with or without 1 μmol/L JQ1 for 24 h was analyzed by Western blot analysis; (H) The protein expression in cells transfected with si-BRD4 for 24 or 48 h was checked by Western blot analysis (left) and quantitatively analyzed (right); (I) The protein expression in MDA-MB-231 cells treated with 1 μmol/L JQ1 for 0–24 h or increasing concentrations of JQ1 for 24 h was checked; (J) sh-Control or sh-ALKBH5 MDA-MB-231 and BT-549 cells were treated with or without 1 μmol/L JQ1 for 24 h. The m 6 A/A ratio of mRNA was checked by LC–MS/MS; (K) sh-Control or sh-ALKBH5 MDA-MB-231 cells were treated with or without 1 μmol/L JQ1 for 24 h. The migration was checked by wound-healing assay (left) and quantitatively analyzed (right), scale bar: 200 μm; (L) sh-Control or sh-ALKBH5 MDA-MB-231 cells were treated with or without 1 μmol/L JQ1 for 24 h. The invasion was checked by Transwell assay (left) and quantitatively analyzed (right), scale bar: 200 μm; (M) Cell proliferation was checked in sh-Control or sh-ALKBH5 MDA-MB-231 cells treated with or without 1 μmol/L JQ1 for 24 h; (N) Cell proliferation of sh-Control or sh-ALKBH5 MDA-MB-231 cells pre-treated with or without 1 μmol/L JQ1 for 12 h and then further treated with an increasing concentration of Dox for 48 h; (O) The protein expression in sh-Control or sh-ALKBH5 MDA-MB-231 cells treated with or without 1 μmol/L JQ1 for 24 h. Data are presented as mean ± SD from three independent experiments. ∗∗ P < 0.01, ns, no significant, by Student's t test (two-side) as compared with the control group.

Article Snippet: BRD4 plasmid was purchased from Addgene (90,331), and the histone deacetylase 2 (HDAC2) plasmid and empty vector were as a gift from Professor Jiemin Wong at the East China Normal University.

Techniques: Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Transfection, Plasmid Preparation, Control, Construct, Dot Blot, Expressing, Western Blot, Migration, Wound Healing Assay, Transwell Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: BRD4 regulates m 6 A of ESPL1 mRNA via interaction with ALKBH5 to modulate breast cancer progression

doi: 10.1016/j.apsb.2024.12.037

Figure Lengend Snippet: BRD4 regulates the mRNA and protein stability of ALKBH5. (A) The mature mRNA of ALKBH5 in cells treated with 1 μmol/L JQ1 for 0–36 h; (B) Nascent transcripts of ALKBH5 in cells treated with or without 1 μmol/L JQ1 for 24 h were analyzed by 4sU incorporation in the newly synthesized transcripts; (C) The relative levels of cytoplasmic versus nuclear ALKBH5 mRNA in MDA-MB-231 cells treated with or without 1 μmol/L JQ1 for 24 h; (D, E) MDA-MB-231 (D) or BT-549 (E) cells were pre-treated with or without 1 μmol/L JQ1 for 24 h and further treated with Act-D for 0–8 h. The mRNA of ALKBH5 was checked by qRT-PCR; (F) MDA-MB-231 cells were pre-transfected with si-NC or si-BRD4 for 24 h and further treated with Act-D for 0–8 h. The mRNA of ALKBH5 was checked by qRT-PCR; (G) The protein of ALKBH5 in MDA-MB-231 cells pre-treated with or without 1 μmol/L JQ1 for 6 h and further treated with CHX for 0–8 h was checked by Western blot analysis (left) and quantitatively analyzed (right); (H) The protein of ALKBH5 in MDA-MB-231 cells pre-transfected with si-NC or si-BRD4 for 24 h and further treated with CHX for 0–8 h; (I) The protein of ALKBH5 in MDA-MB-231 cells pre-treated with or without MG-132 for 90 min and further treated with or without 1 μmol/L JQ1 for 24 h; Data are presented as mean ± SD from three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, ns, no significant, by Student's t test (two-side) as compared with the control group.

Article Snippet: BRD4 plasmid was purchased from Addgene (90,331), and the histone deacetylase 2 (HDAC2) plasmid and empty vector were as a gift from Professor Jiemin Wong at the East China Normal University.

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

Journal: Acta Pharmaceutica Sinica. B

Article Title: BRD4 regulates m 6 A of ESPL1 mRNA via interaction with ALKBH5 to modulate breast cancer progression

doi: 10.1016/j.apsb.2024.12.037

Figure Lengend Snippet: TRIM21 is essential for BRD4-regulated protein stability of ALKBH5. (A) Venn diagram shows substantial and significant overlaps among proteins involved in ubiquitin-mediated proteolysis and proteins bound with ALKBH5 and BRD4 in MDA-MB-231 cells; (B) Protein lysates of MDA-MB-231 cells were immunoprecipitated with BRD4 or ALKBH5 antibodies. The binding with other proteins was checked by immunoprecipitation; (C) MDA-MB-231 cells were treated with 1 μmol/L JQ1 for 24 h. Binding between ALKBH5 and TRIM21 or BRD4 was checked by immunoprecipitation; (D) MDA-MB-231 cells were transfected with si-NC or si-BRD4 for 24 h. Binding between ALKBH5 and TRIM21 or BRD4 was checked by immunoprecipitation; (E) MDA-MB-231 cells were transfected with si-NC or si-ALKBH5 for 24 h. Binding between BRD4 and TRIM21 or ALKBH5 was checked by immunoprecipitation; (F) The protein expression of TRIM21 in cells treated with or without 1 μmol/L JQ1 for 24 h; (G) The protein expression of ALKBH5 in cells transfected with si-NC or si-TRIM21 for 24 h; (H, I) The ubiquitination of ALKBH5 in MDA-MB-231 (H) or BT-549 (I) cells transfected with si-NC or si-TRIM21 for 24 h. An equal amount of ALKBH5 was loaded after IP according to a pre-Western blot; (J) The expression of ALKBH5 in MDA-MB-231 cells pre-transfected with si-NC or si-TRIM21 for 12 h and then further treated with or without 1 μmol/L JQ1 for 24 h; (K) Schematic diagram depicting a set of FLAG-tagged BRD4 expression constructs; (L) 293T cells were transfected with the indicated constructs of FLAG-BRD4, immunoprecipitation with anti-FLAG beads was performed. Blots were probed with the indicated antibodies of ALKBH5 or TRIM21; (M) The expression of ALKBH5 and HDAC2 in cells transfected with vector control or pT-HDAC2 for 24 h; (N) Acetylation of ALKBH5 in cells transfected with vector control or pT-HDAC2 for 24 h was checked by immunoprecipitation; (O) MDA-MB-231 cells were transfected with vector control or pT-HDAC2 for 24 h. The binding between ALKBH5 and BRD4 or between ALKBH5 and TRIM21 was checked by immunoprecipitation; Data are presented as mean ± SD from three independent experiments. ∗∗ P < 0.01, ns, no significant, by Student's t test (two-side).

Article Snippet: BRD4 plasmid was purchased from Addgene (90,331), and the histone deacetylase 2 (HDAC2) plasmid and empty vector were as a gift from Professor Jiemin Wong at the East China Normal University.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Binding Assay, Transfection, Expressing, Western Blot, Construct, Plasmid Preparation, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: BRD4 regulates m 6 A of ESPL1 mRNA via interaction with ALKBH5 to modulate breast cancer progression

doi: 10.1016/j.apsb.2024.12.037

Figure Lengend Snippet: RALY is essential for BRD4-regulated mRNA stability of ALKBH5 . (A) Relative luciferase activity of F-Luc/R-Luc in cells pre-transfected with pmiR-GLO-ALKBH5-3′UTR for 12 h and then further treated with or without 1 μmol/L JQ1 for 24 h, or in cells transfected with pmiR-GLO-ALKBH5-3′UTR together with si-NC or si-BRD4 for 24 h; (B) Venn diagram shows substantial and significant overlaps among the top 15 RBPs with highest binding sites, Z-score and RF-values, which were predicted by RBPmap bioinformatics program ; (C, D) Relative luciferase activity of F-Luc/R-Luc in MDA-MB-231 (C) or BT-549 (D) cells transfected with pmiR-GLO-ALKBH5-3′UTR together with si-NC, si-TARDBP, si-RALY, or si-U2AF2 for 24 h; (E) Relative levels of ALKBH5 mRNA in BC cells transfected with si-NC, si-TARDBP, si-RALY, or si-U2AF2 for 24 h; (F) Protein levels of ALKBH5 in cells transfected with si-NC or si-RALY for 24 h; (G) The mRNA of ALKBH5 in MDA-MB-231 cells pre-transfected with si-NC or si-RALY for 24 h and further treated with Act-D for 0–8 h; (H) Binding between RALY and ALKBH5 mRNA was checked by ChIP-PCR using IgG or RALY antibody; (I, J) MDA-MB-231 cells were pre-transfected with si-NC or si-RALY for 12 h and further treated with or without 1 μmol/L JQ1 for 24 h. The relative mRNA (I) and protein (J) levels of ALKBH5 were checked; (K) MDA-MB-231 cells were pre-transfected with si-NC or si-RALY for 12 h and further treated with or without 1 μmol/L JQ1 for 24 h. Cells were then treated with Act-D for 0–8 h. The mRNA of ALKBH5 was checked by qRT-PCR; (L, M) Cells were treated with or without 1 μmol/L JQ1 for 24 h. The mRNA (L) and protein (M) levels of RALY were checked; (N, O) Cells were treated with or without 1 μmol/L JQ1 for 24 h. Nascent transcripts (N) and precursor mRNA (O) of RALY were analyzed by 4sU incorporation in the newly synthesized transcripts and qPCR, respectively. Data are presented as mean ± SD from three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, ns, no significant, by Student's t test (two-side) as compared with the control group.

Article Snippet: BRD4 plasmid was purchased from Addgene (90,331), and the histone deacetylase 2 (HDAC2) plasmid and empty vector were as a gift from Professor Jiemin Wong at the East China Normal University.

Techniques: Luciferase, Activity Assay, Transfection, Binding Assay, Quantitative RT-PCR, Synthesized, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: BRD4 regulates m 6 A of ESPL1 mRNA via interaction with ALKBH5 to modulate breast cancer progression

doi: 10.1016/j.apsb.2024.12.037

Figure Lengend Snippet: ESPL1 is an effector for BRD4/ALKBH5 axis-regulated cell cycle progression. (A, B) Cells were treated with or without 1 μmol/L JQ1 for 24 h. The mRNA (A) and protein (B) levels of ESPL1 were checked; (C) Cell proliferation of MDA-MB-231 cells pre-transfected with vector control or ESPL1 construct for 12 h were then further treated with or without 1 μmol/L JQ1 for 1–3 days; (D) MDA-MB-231 cells were synchronized at the G1/S transition by a double TdR block and then transfected with vector control or ESPL1 construct for 12 h and then further treated with or without 1 μmol/L JQ1 for 24 h. The cell cycles were analyzed by flow cytometry (FCM); (E) The m 6 A levels of ESPL1 in MDA-MB-231 cells treated with or without 1 μmol/L JQ1 for 24 h were checked by m 6 A-RIP-PCR; (F) m 6 A peaks were decreased in ESPL1 mRNA in JQ1-treated MDA-MB-231 cells from m 6 A RIP-seq data; (G, H) The mRNA (G) and protein (H) expression of ESPL1 in sh-Control or sh-ALKBH5 MDA-MB-231 cells treated with or without 1 μmol/L JQ1 for 24 h; (I) The m 6 A levels of ESPL1 in sh-Control or sh-ALKBH5 MDA-MB-231 cells treated with or without 1 μmol/L JQ1 for 24 h; (J) The predicted m 6 A peaks in ESPL1 mRNA from the m 6 A sites predictor SRAMP; (K) MDA-MB-231 cells were transfected with either wild-type ESPL1 overexpression plasmids or plasmids with mutated predicted m 6 A sites (“A” to “C”) for 24 h. After treating JQ1 for another 24 h, the m 6 A modification levels of ESPL1 mRNA was detected using m 6 A-RIP-qPCR; (L) After transfecting MDA-MB-231 cells with either wild-type ESPL1 overexpression plasmids or plasmids with mutated A4571 sites for 24 h, the m 6 A modification levels of ESPL1 mRNA was detected using m 6 A-RIP-qPCR; (M) MDA-MB-231 cells were treated with or without 1 μmol/L JQ1 for 24 h. The relative binding between ESPL1 mRNA and IGF2BPs was checked by RIP-PCR using antibodies of IGF2BP1/2/3, respectively; (N, O) The protein (N) and mRNA (O) expression of ESPL1 in cells transfected with si-NC or si-IGF2BP3 for 24 h; (P) The mRNA of ESPL1 in MDA-MB-231 cells pre-transfected with si-NC or si-IGF2BP3 for 24 h and further treated with Act-D for 0–8 h; (Q) Proposed model for mechanistic action of ESPL1 as an effector for BRD4/ALKBH5 axis-regulated cell cycle progression. JQ1 inhibits the binding of BRD4 to ALKBH5, leading to a reduction in ALKBH5 ubiquitination levels and an upregulation of ALKBH5 expression. ALKBH5 recognizes and binds to ESPL1 mRNA m 6 A through IGF2BP3, thereby removing the m 6 A modification on ESPL1 mRNA, ultimately resulting in the degradation of ESPL1 mRNA. Data are presented as mean ± SD from three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01, ns, no significant, by Student's t test (two side). Fig. 6 I and J were analyzed by one-way ANOVA followed by Bonferroni test.

Article Snippet: BRD4 plasmid was purchased from Addgene (90,331), and the histone deacetylase 2 (HDAC2) plasmid and empty vector were as a gift from Professor Jiemin Wong at the East China Normal University.

Techniques: Transfection, Plasmid Preparation, Control, Construct, Blocking Assay, Flow Cytometry, Expressing, Over Expression, Modification, Binding Assay, Ubiquitin Proteomics

Journal: Acta Pharmaceutica Sinica. B

Article Title: BRD4 regulates m 6 A of ESPL1 mRNA via interaction with ALKBH5 to modulate breast cancer progression

doi: 10.1016/j.apsb.2024.12.037

Figure Lengend Snippet: BRD4/ALKBH5/ESPL1 axis regulating the progression of BC. (A) The mRNA expression of BRD4 , EPSL1 , and ALKBH5 was measured in MDA-MB-231 LMF3 and F0 xenograft tumor tissues by qRT-PCR; (B) The protein expression of BRD4, EPSL1, and ALKBH5 was measured in MDA-MB-231 LMF3 , BT-549 LMF3 and their F0 xenograft tumor tissues by Western blot analysis; (C) IHC (ALKBH5 and ESPL1)-stained paraffin-embedded sections obtained from patient-derived tumors treated with or without JQ1, scale bar: 100 μm; (D) Tumor growth curves of mice were xenografts implanted with sh-Con and sh-ALKBH5 MDA-MB-231 cells and further treated with vehicle or JQ1 at indicated time intervals; (E–G) Tumor images (E), volumes (F) and weights (G) of xenografts implanted with sh-Con and sh-ALKBH5 MDA-MB-231 cells and further treated with vehicle or JQ1; (H, I) IHC (Ki-67 and ESPL1)-stained paraffin-embedded sections obtained from xenografts (left) and quantitatively analyzed (right), scale bar: 200 μm; (J–L) The relative mRNA expression of ALKBH5 (J), BRD4 (K), and ESPL1 (L) in BC tumor tissue (T) and normal tissue (N) from Oncomine database; (M, N) Correlation of ESPL1 mRNA with ALKBH5 (M) or IGF2BP3 (N) in 880 breast cancer tissues from the TCGA database; (O, P) Overall survival (OS) in patients with high vs . low levels of ALKBH5 (O) or ESPL1 (P) in BC patients plotted by the Kaplan–Meier method; (Q) Proposed model to illustrate the mechanisms of BRD4/ALKBH5/ESPL1 axis-regulated BC progression. BRD4 regulates ALKBH5 expression through two pathways. Firstly, JQ1 inhibition of BRD4 reduces the binding of TRIM21 to ALKBH5, decreasing the ubiquitination of ALKBH5 by TRIM21, which in turn increases the stability of ALKBH5 protein. HDAC2 can also regulate ALKBH5 protein stability by affecting its acetylation. Secondly, inhibiting BRD4 can increase the transcription level of RALY. RALY binds to the 3′ UTR of ALKBH5 mRNA, increasing its stability. Consequently, inhibiting BRD4 upregulates ALKBH5 levels. ALKBH5 recognizes and binds to ESPL1 mRNA m 6 A through IGF2BP3, thereby removing the m 6 A modification on ESPL1 mRNA, ultimately leading to the degradation of ESPL1 mRNA and influencing the progression of breast cancer. Data are presented as mean ± SD from three independent experiments. ∗ P < 0.01, ∗∗ P < 0.05, ns, no significant, by Student's t test (two-side). Fig. 7 F–I was analyzed by one-way ANOVA followed by Bonferroni test.

Article Snippet: BRD4 plasmid was purchased from Addgene (90,331), and the histone deacetylase 2 (HDAC2) plasmid and empty vector were as a gift from Professor Jiemin Wong at the East China Normal University.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Derivative Assay, Inhibition, Binding Assay, Ubiquitin Proteomics, Modification