Journal: Journal of Virology

Article Title: S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

doi: 10.1128/JVI.01707-18

Figure Lengend Snippet: CDK inhibitors regulate the expression of late genes during EBV lytic replication. (A) qPCR of viral RNAs (center) and viral replication levels (right) in HEK293/EBV(WT) cells treated with CDK inhibitors at 48 h after the induction of lytic replication. Cells were transfected with the BZLF1 expression plasmid and were treated with CDK 2/9 inhibitor (CDK2/9i; 500 nM) or alsterpaullone, 2-cyanoethyl (A2CE; 1 μM) as indicated on the schedule (left). Results shown are means ± SDs from three independent experiments. Double asterisks, P < 0.01; n.s., not significant. (B) qPCR analyses of viral RNA levels (center) and viral replication levels (right) in HEK293/EBV(WT) cells treated with CDK inhibitors 48 h after the induction of lytic replication. Cells were transfected with a BZLF1-expressing plasmid and were treated with CDK2/9i (500 nM) or A2CE (1 μM) as indicated on the schedule (left). Results shown are means ± SDs from three independent experiments. Double asterisks, P < 0.01. (C and D) HEK293/EBV(WT) cells were transfected with a TATT-oriLyt-Luc reporter plasmid together with pSV40-Rluc as an internal control and a BZLF1 expression plasmid as indicated. DMSO or a CDK inhibitor was added to the medium at 24 h p.t. (C) Lysates harvested from the cells at 48 h p.t. were subjected to immunoblot analysis with the indicated antibodies. BZLF1 is an IE gene; BMRF1 and BALF2 are E genes; and BRRF2, BALF4 (gB), and BKRF4 are L genes (11, 48, 55, 56). (D) Lysates were also analyzed using luciferase reporter assays. The results are presented as means ± SDs from three independent transfections after normalization to the internal control (Renilla luciferase activity). Double asterisks, P < 0.01. (E) HEK293/EBV(WT) cells were first transfected with BZLF1 expression vectors and then treated with CDK inhibitors. The virus yields were determined by counting GFP-positive Akata(–) cells. The results are shown as means ± SDs from three independent experiments and are relative to the virus yield of BZLF1 with DMSO treatment (infectivity value, 1). Double asterisks, P < 0.01.

Article Snippet: Anti-CDK1 and anti-CDK2 rabbit polyclonal antibodies were obtained from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Western Blot, Luciferase, Activity Assay, Virus, Infection

Journal: Journal of Virology

Article Title: S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

doi: 10.1128/JVI.01707-18

Figure Lengend Snippet: CDK2 is responsible for the phosphorylation-mediated stabilization of BDLF4. (A) The cell cycle of HEK293/FLAG-BDLF4 cells was synchronized using a double thymidine block (61). Cells were harvested at the indicated times after release of the block and were analyzed by immunoblotting using the indicated antibodies. AS, asynchronized; h.p.r., hours postrelease. (B and C) HEK293/FLAG-BDLF4 cells were transfected with plasmids expressing shRNA targeting CDK1 or CDK2 mRNA. Cells were lysed at 48 h p.t. for immunoblotting using the indicated antibodies. (D) HEK293/FLAG-BDLF4 cells were transfected with FLAG-BZLF1 and/or shRNA expression plasmids as indicated, followed by CHX treatment (50 mg/ml). The protein levels of FLAG-BDLF4 and FLAG-BZLF1 (control) were detected using anti-FLAG antibodies. Results are presented as means ± SDs from three independent experiments and are shown relative to the protein levels in the absence of CHX treatment. Asterisks, P < 0.05; n.s., not significant. (E) HEK293/FLAG-BDLF4 cells transfected with shCDK2 were treated with MG132 for 24 h before harvesting. Lysates were analyzed by immunoblotting using the indicated antibodies. (F) (Left) Lytic replication was induced by transfection of 293/EBV(WT) cells with a BZLF1-expressing plasmid, followed by transfection with plasmids carrying shCDK2 at 8 h p.t.; the cells were harvested at 48 h p.t. (Right) Lysates were subjected to immunoblotting using the indicated antibodies.

Article Snippet: Anti-CDK1 and anti-CDK2 rabbit polyclonal antibodies were obtained from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Phospho-proteomics, Blocking Assay, Western Blot, Transfection, Expressing, shRNA, Control, Plasmid Preparation

Journal: Journal of Virology

Article Title: S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

doi: 10.1128/JVI.01707-18

Figure Lengend Snippet: Phosphorylation of BDLF4 by CDK2 complexes enhances L gene expression. (A) In vitro phosphorylation assay showing that BDLF4 protein was phosphorylated by recombinant Cyc A1/CDK2 and Cyc E1/CDK2, but not by Cyc B1/CDK1. Histone H1 served as the control. The phosphorylation assay used ATP-γ-S as the phosphodonor and an anti-thiophosphate ester (anti-ThioP) antibody to detect substrate phosphorylation. Immunoblotting (IB) using an anti-BDLF4 or anti-histone H1 antibody shows the levels of the recombinant protein used for the assay. (B) List of the serine/threonine residues in BDLF4 phosphorylated by Cyc A1/CDK2 and Cyc E1/CDK2. These residues were identified by LC–MS-MS analysis. (C) Schematic representation of the S/T sites mutated in the A×6 and A×4 mutants. (D) HEK293/EBV(dBDLF4) cells were transfected with the pTATT-oriLyt-Luc reporter plasmid together with pSV40-Rluc as an internal control and BZLF1 and BDLF4 (WT or mutant) expression plasmids as indicated. Luciferase activities were measured at 48 h posttransfection. The results are presented as means ± SDs from three independent transfections after normalization against the internal control (Renilla luciferase activity). Lysates were subjected to immunoblotting using the indicated antibodies. Asterisks, P < 0.05. (E) HEK293/EBV(dBDLF4) cells were transfected with BZLF1 and BDLF4 (WT or mutant) expression plasmids as indicated. The viral yields were determined by counting GFP-positive Akata(–) cells. The results are shown as means ± SDs from three independent experiments relative to the viral yield of BZLF1 plus WT BDLF4 (infectivity value, 1). Double asterisks, P < 0.01.

Article Snippet: Anti-CDK1 and anti-CDK2 rabbit polyclonal antibodies were obtained from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Phospho-proteomics, Gene Expression, In Vitro, Recombinant, Control, Western Blot, Liquid Chromatography with Mass Spectroscopy, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Luciferase, Activity Assay, Infection

Journal: Journal of Virology

Article Title: S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

doi: 10.1128/JVI.01707-18

Figure Lengend Snippet: Phosphorylation of BDLA4 at T91 plays an important role in the stabilization of BDLF4 protein. (A) Lysates from HEK293 cells transfected with WT BDLF4 or BDLF4 A mutants were analyzed by immunoblotting using anti-FLAG and anti-GAPDH antibodies. (B) HEK293 cells were first transfected with a BDLF4 WT or T91A mutant expression plasmid and then treated with MG132 or BTZMB. Lysates were analyzed by immunoblotting using the indicated antibodies. (C) HEK293/EBV(dBDLF4) cells were transfected with the pTATT-oriLyt-Luc reporter plasmid, pSV40-Rluc (internal control), and plasmids expressing BZLF1 and BDLF4 (WT, T91A mutant, or T91E mutant) as indicated. Luciferase activities were measured at 48 h posttransfection. The results are presented as means ± SDs from three independent transfections after normalization to the internal control (Renilla luciferase activity) levels. Lysates were subjected to immunoblotting using the indicated antibodies. Asterisks, P < 0.05. (D) HEK293/EBV(dBDLF4) cells were transfected with the pTATT-oriLyt-Luc reporter plasmid, pSV40-Rluc (internal control), and plasmids expressing BZLF1 and BDLF4 (WT or T91A mutant). Luciferase activities in cells treated with CDK inhibitors (CDK2/9i or A2CE) were assayed. The effects of CDK inhibitors on reporter expression are expressed as percentages of decrease from the level of activity for samples treated with DMSO. The results are means ± SDs from three independent experiments. Asterisks, P < 0.05; double asterisks, P < 0.01, respectively. (E) HEK293/EBV(dBDLF4) cells were transfected with BZLF1 and BDLF4 expression vectors as indicated. The viral yields were determined by counting GFP-positive Akata(–) cells. The results are shown as means ± SDs from three independent experiments relative to the viral yield of BZLF1 plus WT BDLF4 (infectivity value, 1). Double asterisks, P < 0.01.

Article Snippet: Anti-CDK1 and anti-CDK2 rabbit polyclonal antibodies were obtained from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Phospho-proteomics, Transfection, Western Blot, Mutagenesis, Expressing, Plasmid Preparation, Control, Luciferase, Activity Assay, Infection

Journal: GeroScience

Article Title: Female baboon adrenal zona fasciculata and zona reticularis regulatory and functional proteins decrease across the life course

doi: 10.1007/s11357-024-01080-9

Figure Lengend Snippet: Antibody antigens, final dilutions, and sources used for immunohistochemical staining of baboon adrenal tissue

Article Snippet: Table 2 Antibody Company Cat # Concentration Negative control Data sheet Citrate synthase (CS) Santa Cruz sc-390693 1:150 Mouse IgG cell sc-390693 (scbt.com) Steroidogenic acute regulatory protein (StAR) Santa Cruz sc-166821 1:2000 Mouse IgG cell sc-166821 (scbt.com) Mechanistic target of rapamycin (mTOR) Santa Cruz sc-517464 1:50 Mouse IgG cell SCBT Cyclin-dependent kinase 4 (CdK4) Santa Cruz sc-166373 1:100 Mouse IgG cell sc-166373 (scbt.com) Cyclin-dependent kinase 1/2 (CdK1,2) Santa Cruz sc-53219 1:50 Mouse IgG cell sc-53219 (scbt.com) Cyclophilin D (CPY40) abcam ab181983 1:500 Normal rabbit serum datasheet_181983 (2).pdf Protein Ki67 (Ki67) Novocastra NCL-ki67-mm1 1:50 Mouse IgG cell Novocastra Ki67 Antibody for IHC Staining (leicabiosystems.com) Nitrotyrosine (NT) Santa Cruz sc-32757 1:500 Mouse IgG cell sc-32757 (scbt.com) Glucocorticoid receptor (GR) Santa Cruz sc-393232 1:50 Mouse IgG cell sc-393232.qxp (scbt.com) Cyclin-dependent kinase inhibitor 1 (P21) Santa Cruz sc-817 1:150 Mouse IgG cell sc-817 (scbt.com) Proliferating cell nuclear antigen (PCNA) Santa Cruz sc-56 1:1000 Mouse IgG cell sc-56 (scbt.com) ATP-synthase (ATP5A) Santa Cruz sc- 136,178 1:200 Mouse IgG cell sc-136178 (scbt.com) Open in a separate window Antibody antigens, final dilutions, and sources used for immunohistochemical staining of baboon adrenal tissue.

Techniques: Immunohistochemical staining, Staining, Concentration Assay, Negative Control, Immunohistochemistry

Journal: Scientific Reports

Article Title: miR-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein

doi: 10.1038/s41598-018-22767-y

Figure Lengend Snippet: miR-3140 targeted CDK2 and EGFR by binding their 3′UTR regions. ( a ) Left, identification of downregulated genes after miR-3140 transfection by a gene expression array. The Venn diagram shows that 228 genes were commonly downregulated (fold change >2) by transfection of miR-3140 in Panc1, MIAPaCa2, and MDA-MB-231 cells. Right, prediction of candidate target genes regulated by miR-3140 via their 3′UTR. The Venn diagram shows that 99 genes were predicted as candidate 3′UTR-targets of miR-3140 by the TargetScan program. ( b ) Western blot analysis of CDK2, CDK6, and EGFR in Panc1 and MIAPaCa2 cells 72 hours after transfection with 10 nmol/L of miR-NC or miR-3140 . ( c ) Luciferase reporter assays. Panc1 cells were transfected with the pmirGLO Dual Luciferase vectors containing wild type (Wt) CDK2 and EGFR or mutant (Mt) 3’UTR target sites of these genes, and after 6 hours, either miR-NC or miR-3140 was additionally transfected. Top, putative binding site of miR-3140 within the 3′UTR of each gene and mutant sequences. Bottom, results of the luciferase assay; * P < 0.05. ( d , e ) Evaluation of the effect of si-CDK2 or si-EGFR . Western blot analysis (top) and cell growth assay (bottom) in indicated cell lines after transfection with 20 nmol/L of negative control siRNA ( si-NC ) or siRNA targeting each gene. ( f ) Evaluation of the effect of miR-3140 in EGFR-TKI-resistant lung cancer cells. Western blot analysis (top) and cell growth assay (bottom) in the indicated cell line after transfection with 10 nmol/L of miR-NC or miR-3140 . Cell growth ratio was assessed with the WST-8 assay based on the relative ratio compared with day 1. Bar, SD for triplicate experiments; * P < 0.05.

Article Snippet: The following primary antibodies were used for immunohistochemistry: an antibody for BRD4 (HPA061646, 1:500) was purchased from Atlas Antibodies (Stockholm, Sweden), BRD3 (A302-368A, 1:500) and CDK2 (IHC-00374, 1:500) antibodies were from Bethyl Laboratories, and EGFR (sc-03-G, 1:200) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Transfection, Gene Expression, Western Blot, Luciferase, Mutagenesis, Growth Assay, Negative Control

Journal: Scientific Reports

Article Title: miR-3140 suppresses tumor cell growth by targeting BRD4 via its coding sequence and downregulates the BRD4-NUT fusion oncoprotein

doi: 10.1038/s41598-018-22767-y

Figure Lengend Snippet: Therapeutic effects of miR-3140 for tumor growth in vivo . ( a ) The experimental schedule for miR-3140 treatment in nude mice which were subcutaneously inoculated with MIAPaCa2 cells. ( b ) The representative image of tumor-bearing nude mice at 23 days after the inoculation of MIAPaCa2 cells. Tumors are denoted by arrowheads. ( c ) Tumor growth curves of xenograft mouse models treated with miR-NC or miR-3140 (n = 5, each). Tumor volume was calculated using the following formula: (shortest diameter) 2 × (longest diameter) × 0.5. Bar, SD for 5 mice; * P < 0.05. ( d ) Expression analysis of miR-3140 in resected tumors. The expression level of miR-3140 was measured by qRT-PCR using the relative ratio normalized based on the expression of RNU6B . Each experiment was performed in duplicate. Bar, SD. ( e ) Representative images of immunohistochemical staining for BRD4, BRD3, CDK2 and EGFR in resected tumors. Scale bar, 50 μm \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$.$$\end{document} . ( f ) Schematic models for the mechanism by which miR-3140 suppresses tumor growth.

Article Snippet: The following primary antibodies were used for immunohistochemistry: an antibody for BRD4 (HPA061646, 1:500) was purchased from Atlas Antibodies (Stockholm, Sweden), BRD3 (A302-368A, 1:500) and CDK2 (IHC-00374, 1:500) antibodies were from Bethyl Laboratories, and EGFR (sc-03-G, 1:200) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining