rabbit cdk9 polyclonal antibody  (Proteintech)

Journal: Nature Communications

Article Title: CDK9 recruits HUWE1 to degrade RARα and offers therapeutic opportunities for cutaneous T-cell lymphoma

doi: 10.1038/s41467-024-54354-3

Figure Lengend Snippet: a HH and Hut78 cells were treated with compounds (2 µM) from inhibitor library (L1200) and cell viability was evaluated using CCK8 assay after treatment for 48 hours. CDK Cyclin-dependent kinase, JAK/STAT Janus kinase-signal transducer and activator of transcription, PI3K Phosphoinositide 3-kinases, mTOR mammalian target of rapamycin complex. b The most effective kinase inhibitors (cell viability < 25%) identified from compound screening for HH and Hut78 cells. c Numbers of effective (red, cell viability < 25%) and ineffective (gray, cell viability \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\geq$$\end{document} ≥ 25%) CDK inhibitors from each different CDK subfamilies were shown. d IC 50 curve of five representative CDK9 inhibitors, AT7519, Dinaciclib, Flavopiridol, SNS-032 and P276-00 in HH and Hut78 cells. Cell viability was counted by trypan blue staining. Results represent biologically independent experiments of n = 3. e – h Nude mice were subcutaneously injected with HH cells and randomly divided into Vehicle, Flavopiridol and SNS-032 groups (n = 8). Tumor volumes were measured at different time points ( e , g ). At 17 days after subcutaneous injection, tumors were harvested and weighed ( f , h ). i Western blot analysis of indicated proteins from Hut78 cells infected with lentivirus encompassing shNC, shCDK9-1 or shCDK9-2. j Growth curve of Hut78 cells upon CDK9 depletion. Cell number was counted by trypan blue. Experiments were performed in triplicate and repeated twice with similar results. k Western blot analysis of indicated proteins from HH cells infected with lentivirus encompassing shNC, shCDK9-1, or shCDK9-2. l Growth curve of HH cells upon CDK9 depletion. Cell number was counted by trypan blue. Experiments were performed in triplicate and repeated twice with similar results. m , n HH cells were infected with shNC, shCDK9-1, or shCDK9-2 lentiviruses, and subcutaneously injected into nude mice (n = 6). Tumor volumes were measured at different time points ( m ). At 12 days after subcutaneous injection, tumors were harvested and weighed ( n ). Data are presented as mean ± SEM. Unpaired, two-tailed Student’s t -test. Source data are provided as a file. i , k n = 3, independent experiments, a representative example is shown. The samples derive from the same experiment each but different gels for CDK9 and another for β-actin were processed in parallel. Band intensities were analyzed and compared using Image J. Relative densitometric values are provided below the blot images.

Article Snippet: Sections were stained using the polyclonal antibodies CDK9 (Cell Signaling Technology, 2316) or CD25 (AF7224, Beyotime).

Techniques: CCK-8 Assay, Staining, Injection, Western Blot, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: CDK9 recruits HUWE1 to degrade RARα and offers therapeutic opportunities for cutaneous T-cell lymphoma

doi: 10.1038/s41467-024-54354-3

Figure Lengend Snippet: a Immunohistochemistry (IHC) staining of CDK9 of tissues collected from healthy donors (Normal) and CTCL patients. b Immunofluorescent staining of tissue sections from Normal and CTCL patients using antibodies against CD4 and CDK9. DAPI (4’,6-diamidino-2-phenylindole) was used for nucleus staining. c Western blot analysis of samples from healthy donors (Normal) and CTCL cell lines using indicated antibodies. n = 3, independent experiments, a representative example is shown. Band intensities were analyzed and compared using Image J. Relative densitometric values are provided below the blot images. d Differential expression of CDK9 in patients with CTCL from the indicated datasets. NLCT non large-cell transformation. P values were calculated by one-tailed t test. e UMAP projection of CTCL dataset GSE165623 including cells from skin, blood and lymph node across a patient with advanced MF. f Dot plot of CTCL dataset GSE165623 showing the average expression levels and cell expression proportions of selected cell lineage markers. g Violin plots of T cell clusters in CTCL datasets GSE165623 and GSE171811 exhibiting the specific expression of CDK9 and proliferating markers. CDK9 high cluster number was labeled in red. h UMAP projections of three cell cycle phases in CTCL datasets GSE165623, GSE171811, and GSE128531 respectively, T cell clusters were labeled using the bold black text and CDK9 high cluster was indicated by the dotted oval (top panel). Percentage of cells in each phase of cell cycle was shown, with CDK9 high cluster number labeled in red (bottom panel). i UMAP plots of T cells from GSE165623 and GSE171811 datasets, cells with the top expanded TCR α or β chain CDRs amino acid sequence per patient are colored in red (malignant), the polyclonal α or β TCRs are labeled in blue (polyclonal), and cells without detectable TCR are displayed in gray (remaining). Cell distribution was presented with contour graphs and summarized in the accompanying bar chart. The CDK9 high cluster was labeled in red (right panel). j UMAP representation split by tissues of T cells from GSE165623. Malignant T cells from skin, blood, and lymph node are colored by dark cyan, red, and purple. CDK9 high cluster number was labeled using the bold black text and cells were indicated by the dotted oval.

Article Snippet: Sections were stained using the polyclonal antibodies CDK9 (Cell Signaling Technology, 2316) or CD25 (AF7224, Beyotime).

Techniques: Immunohistochemistry, Staining, Western Blot, Quantitative Proteomics, Transformation Assay, One-tailed Test, Expressing, Labeling, Sequencing

Journal: Nature Communications

Article Title: CDK9 recruits HUWE1 to degrade RARα and offers therapeutic opportunities for cutaneous T-cell lymphoma

doi: 10.1038/s41467-024-54354-3

Figure Lengend Snippet: a RNA-seq analysis of Hut78 cells treated with Flavopiridol or knocked down CDK9 expression with shCDK9. The Venn diagram showed the genes differentially expressed in each group. b MOLT-4 and HH cells were treated with THAL-SNS-032 and western blots were conducted. c HH cells were treated with DMSO or candidate PROTACs. Cell viability was measured using the CCK8. d Chemical structure of 23 (GT-02897). e Cell viability of Hut78 cells treated with DMSO or 19, 23, 24, 25, 26, and 27 . Viability was counted by trypan blue. P value was calculated by two-tailed Student’s t -test. f Western blot analysis of indicated proteins in Hut78 cells treated with DMSO or 23, 24, 25, and 26 . g , Western blot analysis of CDK9 in Hut78 cells upon THAL-SNS-032 or 23 (GT-02897) treatment. h 293T cells were treated with DMSO or 23 (GT-02897). Viability was counted by trypan blue. i Hut78 were treated with 23 (GT-02897) in the presence or absence of MG132 followed by Western blot analysis. j Hut78 cells were pre-exposed to SNS-032 or PHA-767491 followed by treatment with 23 (GT-02897). Then CDK9 protein was analyzed by Western blot. k Quantitative proteomic analysis and volcano plot of proteins that were differently regulated in Hut78 cells upon 23 (GT-02897) treatment. CDK9 was labeled. P value was derived by Wilcoxon rank-sum test. l Hut78 cells were treated with 23 (GT-02897) and Q-PCR analysis was performed for CDK9 (55 kDa) and CDK9 (42 kDa). P value was calculated by two-tailed Student’s t -test. m The regulated proteins in ( k ) were annotated and analyzed for the most significantly enriched pathways using Metascape. n – r Western blot analysis of indicated proteins in Hut78 cells upon 23 (GT-02897) treatment. s Hut78 cells were treated with different doses of 23 (GT-02897) for 10 h and DC 50 was calculated. Results in ( e , h , s ) represent biologically independent experiments of n = 3. Results in ( l ), n = 4 for CDK9 (55 kDa) and n = 3 for CDK9 (42 kDa). Data are presented as mean ± SEM. Source data are provided as a file. b , f , g , i , j , n – r n = 3, independent experiments, a representative example is shown. The samples in ( f ) derive from the same experiment but different gels for CDK9, β-actin, another for CDK7, another for CDK2 and another for CDK6 were processed in parallel. Band intensities were analyzed and compared using ImageJ. Relative densitometric values are provided below the blot images.

Article Snippet: Sections were stained using the polyclonal antibodies CDK9 (Cell Signaling Technology, 2316) or CD25 (AF7224, Beyotime).

Techniques: RNA Sequencing, Expressing, Western Blot, Two Tailed Test, Labeling, Derivative Assay

Journal: Nature Communications

Article Title: CDK9 recruits HUWE1 to degrade RARα and offers therapeutic opportunities for cutaneous T-cell lymphoma

doi: 10.1038/s41467-024-54354-3

Figure Lengend Snippet: a – c NSG mice were subcutaneously injected with Hut78 cells (8 × 10 6 cells for each mouse) and randomly divided into two groups receiving intraperitoneal injection of Vehicle or 23 (GT-02897) (n = 4). Tumor volumes were measured at different time points ( a ). At 14 days after subcutaneous injection, tumors were harvested and weighed ( b ), followed by IHC staining of human CDK9 ( c ). P value was calculated by two-tailed Student’s t -test. d , e NSG mice were subcutaneously injected with Hut78 cells (8 × 10 6 cells for each mouse) and randomly divided into two groups receiving subcutaneously injection of Vehicle or 23 (GT-02897) (n = 5). Tumor volumes were measured at different time points ( d ) and survival curves were shown ( e ). P value of tumor volumes was calculated by two-tailed Student’s t -test, P value of survival curve was calculated by log-rank test. f – h NSG mice were subcutaneously injected with Hut78 cells introduced with shNC, shCDK9-1 or shCDK9-2 (8 × 10 6 cells for each mouse) and treated with 23 (GT-02897) (n = 6). Tumor volumes were measured at different time points ( f ). At 12 days after subcutaneous injection, tumors were harvested and weighed ( g , h ). P value was calculated by two-tailed Student’s t -test. Data are presented as mean ± SEM. Source data are provided as a file.

Article Snippet: Sections were stained using the polyclonal antibodies CDK9 (Cell Signaling Technology, 2316) or CD25 (AF7224, Beyotime).

Techniques: Injection, Immunohistochemistry, Two Tailed Test

Journal: Nature Communications

Article Title: CDK9 recruits HUWE1 to degrade RARα and offers therapeutic opportunities for cutaneous T-cell lymphoma

doi: 10.1038/s41467-024-54354-3

Figure Lengend Snippet: a Quantitative proteomic analysis and volcano plot of Hut78 cell proteins upon 23 (GT-02897) treatment, with P values derived from Wilcoxon rank-sum test. b Western blotting of indicated proteins in Hut78 under 23 (GT-02897) treatment. Western blot analysis of indicated proteins in HH and Hut78 ( c ) or 293T ( d ) cells after CDK9 depletion via lentiviruses. e A doxycycline (Dox)-inducible knockdown system was applied in HH and Hut78 cells, followed by Western blotting post Dox treatment. f Volcano plot revealing differentially expressed genes in Hut78 post CDK9 depletion, with P values derived by Wilcoxon rank-sum test. g Q-PCR assessment of CDK9 and RARA levels in Hut78 cells after CDK9 depletion. h Western blot analysis of proteins in Hut78 after CDK9 depletion, with or without MG132 treatment. i 293T cells were co-transfected with RARα and CDK9 constructs, followed by co-immunoprecipitation (Co-IP) and Western blot analysis. j GST-tagged CDK9 was incubated with HA-RARα from the WCL of 293T cells transfected with 3 × HA-tagged RARα, followed by GST pulldown and Western blot analysis. WCL: whole cell lysate. k Schematic diagram of full-length CDK9 protein (top). GST pulldown assay was performed using GST-tagged CDK9 and WCL of 293T cells transfected with 3 × HA-tagged RARα. l Lentiviral infection of Hut78 with shNC or shCDK9-1, followed by transfection of EV, 3 × Flag-tagged WT, T186A, and S347A CDK9, and Western blotting of indicated proteins. m Western blotting of indicated proteins in Hut78 post CDK9 depletion or Flavopiridol treatment. n Q-PCR analysis of RARA in Hut78 cells treated with Flavopiridol. o Schematic diagram of full-length RARα protein (top). Co-transfection of 3×Flag-tagged CDK9 with full-length or mutant RARα constructs in 293T, followed by Co-IP and immunoblotting (bottom). Results in ( g , n ) represent biologically independent experiments of n = 3, with P values calculated by two-tailed Student’s t -test. Data are presented as mean ± SEM, with source data provided as a file. b – e , h – m , o n = 3, independent experiments, a representative example is shown. The samples derive from the same experiment each but different gels for CDK9, β-actin and another for RARα ( b , d , e , h ), for RARα, β-actin and another for CDK9 ( c ), for HA-RARα, β-actin and another for Flag-CDK9 ( i ), for RARα, β-actin and another for Flag-CDK9 ( l ) were processed in parallel. Band intensities in ( b – e , h , l , m ) were analyzed and compared using Image J. Relative densitometric values are provided below the blot images.

Article Snippet: Sections were stained using the polyclonal antibodies CDK9 (Cell Signaling Technology, 2316) or CD25 (AF7224, Beyotime).

Techniques: Derivative Assay, Western Blot, Knockdown, Transfection, Construct, Immunoprecipitation, Co-Immunoprecipitation Assay, Incubation, GST Pulldown Assay, Infection, Cotransfection, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: CDK9 recruits HUWE1 to degrade RARα and offers therapeutic opportunities for cutaneous T-cell lymphoma

doi: 10.1038/s41467-024-54354-3

Figure Lengend Snippet: a Hut78 cells were transfected with 3 × Flag-tagged WT, T186A, or S347E CDK9, followed by Co-IP with anti-Flag antibody. Mouse IgG was used as negative control. Samples were then analyzed via LC-MS/MS and E3 ligases interacting with 3 × Flag-tagged WT, T186A, or S347E CDK9 were listed. b Bacterially expressed GST-tagged CDK9 was incubated with Hut78 WCL, followed by GST pulldown and Western blot analysis. WCL: whole cell lysate. c – e Western blot analysis of indicated proteins in Hut78 ( c ) and 293T ( d ) cells with HUWE1 depletion, as well as in wild-type (WT) and HUWE1 knockout (KO) MEF cells ( e ). f Hut78 cells overexpressing RARα were infected with shNC or shHUWE1 lentiviruses. After 6 h of treatment with MG132 (10 µM) or DMSO, Co-IP with anti-RARα or mouse IgG was conducted, followed by immunoblotting. g In 293T cells transfected with 3 × HA-tagged RARα, Myc-tagged ubiquitin, and HUWE1, Co-IP and immunoblotting were conducted after MG132 (10 µM) or DMSO treatment. h Schematic of RARα ubiquitination K sites (left) and protein structure of RARα ligand binding domain (LBD) (in white) bound to ATRA (in blue) (PDB ID: 3A9E) (right). i Tandem mass spectrum of a RARα-derived peptide confirmed ubiquitin conjugation at residue K360. j Myc-tagged ubiquitin was co-transfected with wild-type or mutant RARα (HA-RARα WT , HA-RARα K244R , and HA-RARα K360R ) into 293T cells with or without infection of HUWE1. Following 6 h of MG132 (10 µM) treatment, Co-IP and immunoblotting were performed. k , Myc-tagged ubiquitin was co-transfected with wild-type or mutant RARα with deletion of the Hinge domain (HA-ΔH-RARα WT , HA-ΔH-RARα K244R , and HA-ΔH-RARα K360R ) into 293T cells with or without infection of HUWE1. Following 6 h of MG132 (10 µM) treatment, Co-IP and immunoblotting were performed. l Western blot analysis of indicated proteins in wild-type (WT) and HUWE1 knockout (KO) MEF cells with or without infection of 3×Flag-tagged CDK9. b – g and j – l n = 3, independent experiments, a representative example is shown. The samples derive from the same experiment each but different gels for RARα, β-actin, another for HUWE1 and another for GST-CDK9, GST ( b ), for RARα, β-actin and another for HUWE1 ( c – e ), for RARα, β-actin and another for Ub ( f ), for HA-RARα, β-actin, another for HUWE1 and another for Myc ( g , j , k ), for RARα, β-actin, another for Flag-CDK9 and another for HUWE1 ( l ) were processed in parallel. Band intensities in ( c – e , l ) were analyzed and compared using Image J. Relative densitometric values are provided below the blot images.

Article Snippet: Sections were stained using the polyclonal antibodies CDK9 (Cell Signaling Technology, 2316) or CD25 (AF7224, Beyotime).

Techniques: Transfection, Co-Immunoprecipitation Assay, Negative Control, Liquid Chromatography with Mass Spectroscopy, Incubation, Western Blot, Knock-Out, Infection, Ubiquitin Proteomics, Ligand Binding Assay, Derivative Assay, Conjugation Assay, Residue, Mutagenesis

Journal: Nature Communications

Article Title: CDK9 recruits HUWE1 to degrade RARα and offers therapeutic opportunities for cutaneous T-cell lymphoma

doi: 10.1038/s41467-024-54354-3

Figure Lengend Snippet: a Western blotting in Hut78 cells upon HA-RARα overexpression. b Growth curve of Hut78 cells infected with EV or HA-RARα lentivirus. Cell number was counted by trypan blue. c A doxycycline (Dox)-inducible knockdown system (shNC or shRARα) was introduced into Hut78 cells via lentiviruses, followed by Dox administration and Western blot analysis. d Growth curves of Hut78 cells with Dox-inducible shNC or shRARα post-Dox treatment, with cell counts via trypan blue. e Western blot analysis of CDK9 and RARα in CDK9-depleted Hut78 cells with or without RARα depletion. f Growth curve of control and CDK9-depleted Hut78 cells infected with or without shRARα lentivirus. Cell number was counted by trypan blue. g Hut78 cells were treated with ATRA or DMSO. Viability and cell number were measured by trypan blue. h RNA-seq analysis of HH and Hut78 cells upon ATRA treatment, with a Venn diagram showing common differentially expressed genes (left), and enriched pathways analyzed via Metascape (right). i Heatmap of regulated genes upon ATRA treatment in HH and Hut78 cells. j Schematic of CTCL cell differentiation into T cell lineages after ATRA treatment. k Q-PCR analysis of indicated genes in Hut78 cells after ATRA treatment. l Flow cytometry analysis of CD25, CD5 and CD7 expression on HH (top) and Hut78 (bottom) cells upon ATRA or DMSO treatment. m Growth curves of Hut78 cells infected with shNC or shCDK9 lentiviruses upon ATRA or DMSO treatment. Cell number was counted by trypan blue. n Hut78 cells were treated with ATRA and GT-02897 alone or combined and the cell number was counted by trypan blue. o – q NSG mice were subcutaneously injected with Hut78 cells and randomly divided into four groups (Vehicle, GT-02897, ATRA or ATRA + GT-02897, n = 10). Tumor volumes were measured over time ( o ).Tumors were harvested at 21 days, weighed ( p ), and analyzed by IHC for human CD25 ( q ). Results in ( b , d , f , g , k , m , n ) represent biologically independent experiments of n = 3, with P values calculated by two-tailed Student’s t -test. Data are presented as mean ± SEM. Source data are provided as a file. a , c , e n = 3, independent experiments, a representative example is shown. The samples in ( e ) derive from the same experiment but different gels for CDK9, β-actin, and another for RARα were processed in parallel. Band intensities in ( a , c , e ) were analyzed and compared using Image J. Relative densitometric values are provided below the blot images.

Article Snippet: Sections were stained using the polyclonal antibodies CDK9 (Cell Signaling Technology, 2316) or CD25 (AF7224, Beyotime).

Techniques: Western Blot, Over Expression, Infection, Knockdown, Control, RNA Sequencing, Cell Differentiation, Flow Cytometry, Expressing, Injection, Two Tailed Test

Journal: iScience

Article Title: Evolutionary analysis reveals the role of a non-catalytic domain of peptidyl arginine deiminase 2 in transcriptional regulation

doi: 10.1016/j.isci.2024.109584

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CDK9 (D-7) , Santa Cruz Biot. , SC-13130.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Clone Assay, Plasmid Preparation, Mutagenesis, Software

Journal: Nature communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation.

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: Fig. 4 | Real-time visualization of protein degradation and mechanism of DbTACs. a Live-cell imaging was performed to visualize the real-time localization of CDK9 in HEK293T cells and to track the decrease in CDK9 after treatment with DbTACs-26 Å for 6 h. The scale bars, 40 μm. b SEC-HPLC analysis of retention time of DbTACs-26 Å after incubation with human recombinant CDK9 or CRBN protein or both. c Molecular docking sites of the ternary complex in an all-atom model. SPR sensorgrams were employed to monitor the interaction between e DbTACs-26 Å (binary complexes) or d DbTACs-26 Å, f DbTACs-8 Å, and g DbTACs-57 Å

Article Snippet: Primary antibodies used in this study were rabbit GAPDH polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 10494-1-AP, 1:10000), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:1000), mouse CDK1/2 (AN21.2) monoclonal antibody (Santa Cruz Biotechnology, sc-53219, 1:250), mouse CDK6 antibody (ProteintechGroup, Rosemont, IL, USA, 66278-1-Ig, 1:1000), rabbit ERG polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 14356-1- AP, 1:1000), rabbit HPK1 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 23950-1-AP, 1:1000).

Techniques: Live Cell Imaging, Incubation, Recombinant

Journal: Nature communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation.

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: Fig. 7 | Design, preparation, characterization, and efficacy of Abs-DbTACs formed using antibody as POI ligand. a Strategy for designing Abs-DbTACs using CDK9 antibody as the POI ligand. b Self-assembly process of Abs-DbTACs was analyzed by agarose gel electrophoresis. The preparation of Abs-DbTACs was verified by c UV‒visible spectra and d SEC-HPLC. e WB analysis of the targeted CDK9 degradation ability of Abs-DbTACs at different concentrations in MOLM13

Article Snippet: Primary antibodies used in this study were rabbit GAPDH polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 10494-1-AP, 1:10000), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:1000), mouse CDK1/2 (AN21.2) monoclonal antibody (Santa Cruz Biotechnology, sc-53219, 1:250), mouse CDK6 antibody (ProteintechGroup, Rosemont, IL, USA, 66278-1-Ig, 1:1000), rabbit ERG polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 14356-1- AP, 1:1000), rabbit HPK1 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 23950-1-AP, 1:1000).

Techniques: Agarose Gel Electrophoresis

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 1. Different CDK9 staining intensities and H&E staining of endometrial cancer tissues. According to the CDK9 staining in the tumor samples, the staining patterns were divided into 5 groups: i) l<10% positive cells (1+); ii) 10‑25% positive cells (2+); iii) 26‑50% positive cells (3+); iv) 51‑75% positive cells (4+); v) >75% positive cells (5+). (Original magnification, x400). CDK9, cyclin‑dependent kinase 9; H&E, hematoxylin and eosin.

Article Snippet: Thereafter, the slides were sealed with goat serum for 1 h, and then polyclonal rabbit antibodies against human CDK9 (cat. no. 2316; 1:50 dilution in 1% bovine serum albumin; Cell Signaling Technology, Inc.) were added and incubated overnight.

Techniques: Staining

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 3. CDK9 expression in endometrial cancer cell lines. (A) Expression levels of CDK9 in endometrial cancer cell lines (AN3CA, ARK‑2, HEC‑1A, HEC‑1B, lshikawa, RL95‑2 and SPAC1S) as determined by western blotting. (B) Relative expression of CDK9 and α‑tubulin in the endometrial cancer cell lines. CDK9, cyclin‑dependent kinase 9.

Article Snippet: Thereafter, the slides were sealed with goat serum for 1 h, and then polyclonal rabbit antibodies against human CDK9 (cat. no. 2316; 1:50 dilution in 1% bovine serum albumin; Cell Signaling Technology, Inc.) were added and incubated overnight.

Techniques: Expressing, Western Blot

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 2. Higher expression of CDK9 is present in metastatic and recurrent endometrial cancer tissues compared with that found in the patient matched primary tumors and CDK9 is correlated with poor patient prognosis. (A) Distribution of CDK9 immunohistochemical staining scores among primary, metastatic, and recurrent endometrial cancer tissues. (B and C) Correlation between expression of CDK9 in the primary endometrial cancer tissues (Low, CDK9 staining ≤2+; High, CDK9 staining ≥3+) and PFS (B) or OS (C) in endometrial cancer patients by Kaplan‑Meier survival curve analysis. CDK9, cyclin‑dependent kinase 9; PFS, progression‑free survival; OS, overall survival.

Article Snippet: Thereafter, the slides were sealed with goat serum for 1 h, and then polyclonal rabbit antibodies against human CDK9 (cat. no. 2316; 1:50 dilution in 1% bovine serum albumin; Cell Signaling Technology, Inc.) were added and incubated overnight.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 5. CDK9 inhibitor reduces endometrial cancer cell proliferation by suppressing transcription elongation and inducing apoptosis in endometrial cancer cells. (A and B) Relative cell viability of AN3CA and SPAC1S cells after exposure to different concentrations of the CDK9 inhibitor LDC067 for 5 days. **P<0.01 compared with the lowest concentration group (1x10‑3 µM). (C and D) Expression levels of CDK9 and related signaling pathway proteins involved in transcription and apoptosis after treatment with LDC067 in cells by western blot analysis. CDK9, cyclin‑dependent kinase 9; Mcl‑1, myeloid cell leukemia‑1; Bax, proapoptotic protein BCL2 associated X, apoptosis regulator; PARP, poly(ADP‑ribose) polymerase.

Article Snippet: Thereafter, the slides were sealed with goat serum for 1 h, and then polyclonal rabbit antibodies against human CDK9 (cat. no. 2316; 1:50 dilution in 1% bovine serum albumin; Cell Signaling Technology, Inc.) were added and incubated overnight.

Techniques: Concentration Assay, Expressing, Western Blot

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 4. CDK9 knockdown by siRNA transfection suppresses endometrial cancer cell proliferation. (A and B) MTT assay revealed significant dose‑dependent inhibition of cell proliferation after CDK9 siRNA treatment. **P<0.01 compared with the cell only control group. (C and D) Expression levels of CDK9 and related signaling pathway proteins involved in transcription and apoptosis after transfection of CDK9 siRNA and nonspecific siRNA (NC siRNA) in AN3CA and SPAC1S cell lines by western blot analysis. CDK9, cyclin‑dependent kinase 9; Mcl‑1, myeloid cell leukemia‑1; Bax, proapoptotic protein BCL2 associated X, apoptosis regulator.

Article Snippet: Thereafter, the slides were sealed with goat serum for 1 h, and then polyclonal rabbit antibodies against human CDK9 (cat. no. 2316; 1:50 dilution in 1% bovine serum albumin; Cell Signaling Technology, Inc.) were added and incubated overnight.

Techniques: Knockdown, Transfection, MTT Assay, Inhibition, Control, Expressing, Western Blot

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 7. Inhibition of CDK9 reduces endometrial cancer cell migration. (A and B) Representative images of AN3CA and SPAC1S cell migration after CDK9 inhibitor LDC067 treatment for 0, 24, and 48 h. (C and D) Quantification of cell migration distance of AN3CA and SPAC1S cells after LDC067 treatment. **P<0.01 compared with the Cell only group. CDK9, cyclin‑dependent kinase 9.

Article Snippet: Thereafter, the slides were sealed with goat serum for 1 h, and then polyclonal rabbit antibodies against human CDK9 (cat. no. 2316; 1:50 dilution in 1% bovine serum albumin; Cell Signaling Technology, Inc.) were added and incubated overnight.

Techniques: Inhibition, Migration

Journal: Oncology reports

Article Title: Targeting CDK9: A novel biomarker in the treatment of endometrial cancer.

doi: 10.3892/or.2020.7746

Figure Lengend Snippet: Figure 6. Inhibition of CDK9 suppresses endometrial cancer cell colony formation. (A) Representative images of endometrial cancer cell colony formation after incubation with different concentrations of LDC067 (0, 2.5, 5.0, and 10 µM) for 14 days. (B and C) Quantification of clonogenicity formation of AN3CA (B) and SPAC1S (C) cells after LDC067 treatment. **P<0.01 compared with the Cell only group. CDK9, cyclin‑dependent kinase 9.

Article Snippet: Thereafter, the slides were sealed with goat serum for 1 h, and then polyclonal rabbit antibodies against human CDK9 (cat. no. 2316; 1:50 dilution in 1% bovine serum albumin; Cell Signaling Technology, Inc.) were added and incubated overnight.

Techniques: Inhibition, Incubation

Journal: Frontiers in Molecular Biosciences

Article Title: DNA Damage Induces Dynamic Associations of BRD4/P-TEFb With Chromatin and Modulates Gene Transcription in a BRD4-Dependent and -Independent Manner

doi: 10.3389/fmolb.2020.618088

Figure Lengend Snippet: Dynamic associations of BRD4, P-TEFb, and RNAPII with chromatin after UV treatment. Nuclear soluble fraction and chromatin fraction were isolated from UV-treated cells at different time points (0, 0.5, 1, and 2 h). Nuclear soluble fraction and chromatin fraction were lysed in SDS-loading buffer and analyzed by Western blot to determine the amount of BRD4, Cyclin T1, and CDK9 remaining in nuclear fraction and chromatin fraction, and the dynamics of total RNAPII and Ser5 and Ser2 phosphorylation from both nuclear soluble fraction and chromatin fraction. An anti-histone H3 antibody was used as a loading control for chromatin fraction. An anti-TFIIB antibody was used as the loading control for both nuclear soluble fraction and chromatin fraction.

Article Snippet: The following antibodies were used in this study: polyclonal anti-rabbit against BRD4 (ab75898), polyclonal anti-rabbit against RNA polymerase II (Santa Cruz Biotechnologies, sc-9001), polyclonal anti-rabbit against RNA polymerase II CTD repeat YSPTSPS (phospho S5) (ab5131), polyclonal anti-rabbit against RNA polymerase II CTD repeat YSPTSPS (phospho S2) (ab5095), polyclonal anti-rabbit against CDK9 (Santa Cruz Biotechnologies, sc-8338), polyclonal anti-rabbit against Cyclin T1 (Santa Cruz Biotechnologies, sc-10750), polyclonal anti-rabbit against HEXIM1 (ab25388), polyclonal anti-rabbit against Histone H3 (ab1791), and monoclonal anti-mouse against TFIIB (ab819).

Techniques: Isolation, Western Blot, Phospho-proteomics, Control

Journal: Frontiers in Molecular Biosciences

Article Title: DNA Damage Induces Dynamic Associations of BRD4/P-TEFb With Chromatin and Modulates Gene Transcription in a BRD4-Dependent and -Independent Manner

doi: 10.3389/fmolb.2020.618088

Figure Lengend Snippet: BRD4 knockdown results in differential associations of P-TEFb, HEXIM1, and Ser2 phosphorylation of RNAPII CTD with chromatin. (A) Western blot analysis of BRD4 and P-TEFb levels in NEs from HeLa-S3 cells stably expressing BRD4 shRNA or empty vector. TFIIB was used as a loading control. (B) Nuclear soluble fraction and chromatin fraction were isolated from control cells or BRD4-depleted cells treated with UV at different time points (0, 0.5, 1, and 2 h) and immunoblotted with antibodies (anti-CyclinT1, anti-CDK9, anti-HEXIM1, anti-RNAPII, anti-RNAPII phosphorylated at Ser5, anti-RNAPII phosphorylated at Ser2, anti-histone H3, and anti-TFIIB), as shown on the left. An anti-histone H3 antibody was used as a loading control for chromatin fraction. An anti-TFIIB antibody was used as the loading control for both nuclear soluble fraction and chromatin fraction.

Article Snippet: The following antibodies were used in this study: polyclonal anti-rabbit against BRD4 (ab75898), polyclonal anti-rabbit against RNA polymerase II (Santa Cruz Biotechnologies, sc-9001), polyclonal anti-rabbit against RNA polymerase II CTD repeat YSPTSPS (phospho S5) (ab5131), polyclonal anti-rabbit against RNA polymerase II CTD repeat YSPTSPS (phospho S2) (ab5095), polyclonal anti-rabbit against CDK9 (Santa Cruz Biotechnologies, sc-8338), polyclonal anti-rabbit against Cyclin T1 (Santa Cruz Biotechnologies, sc-10750), polyclonal anti-rabbit against HEXIM1 (ab25388), polyclonal anti-rabbit against Histone H3 (ab1791), and monoclonal anti-mouse against TFIIB (ab819).

Techniques: Knockdown, Phospho-proteomics, Western Blot, Stable Transfection, Expressing, shRNA, Plasmid Preparation, Control, Isolation

Journal: Frontiers in Molecular Biosciences

Article Title: DNA Damage Induces Dynamic Associations of BRD4/P-TEFb With Chromatin and Modulates Gene Transcription in a BRD4-Dependent and -Independent Manner

doi: 10.3389/fmolb.2020.618088

Figure Lengend Snippet: Dynamic recruitments of BRD4, P-TEFb, and RNAPII onto gene promoter and gene body of BRD4 positively regulated gene GATA3 after UV stress. (A) BRD4 knockdown efficiency in cells at 0, 0.5, and 2 h after UV treatment. (B) Representative genomic loci showing BRD4 binding at GATA3 locus. (C) RT-PCR analysis of UV-upregulated gene GATA3 expression after BRD4 knockdown. (D) ChIP-qPCR analysis of isolated DNA associated with BRD4 at both promoter and gene body of GATA3 in control and BRD4-depleted HeLa cells after UV treatment. (E) ChIP-qPCR analysis of isolated DNA associated with Ser5-CTD of RNAPII at both promoter and gene body of GATA3 in control and BRD4-depleted HeLa cells after UV treatment. (F) ChIP-qPCR analysis of isolated DNA associated with CDK9 at both promoter and gene body of GATA3 in control and BRD4-depleted HeLa cells after UV treatment. (G) ChIP-qPCR analysis of isolated DNA associated with RNAPII at both promoter and gene body of GATA3 in control and BRD4-depleted HeLa cells after UV treatment. (H) ChIP-qPCR analysis of isolated DNA associated with Ser2-CTD of RNAPII at both promoter and gene body of GATA3 in control and BRD4-depleted HeLa cells after UV treatment. The data in (C) to (H) are reported as mean values ± SD with the indicated significance by using Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: The following antibodies were used in this study: polyclonal anti-rabbit against BRD4 (ab75898), polyclonal anti-rabbit against RNA polymerase II (Santa Cruz Biotechnologies, sc-9001), polyclonal anti-rabbit against RNA polymerase II CTD repeat YSPTSPS (phospho S5) (ab5131), polyclonal anti-rabbit against RNA polymerase II CTD repeat YSPTSPS (phospho S2) (ab5095), polyclonal anti-rabbit against CDK9 (Santa Cruz Biotechnologies, sc-8338), polyclonal anti-rabbit against Cyclin T1 (Santa Cruz Biotechnologies, sc-10750), polyclonal anti-rabbit against HEXIM1 (ab25388), polyclonal anti-rabbit against Histone H3 (ab1791), and monoclonal anti-mouse against TFIIB (ab819).

Techniques: Knockdown, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, ChIP-qPCR, Isolation, Control

Journal: Frontiers in Molecular Biosciences

Article Title: DNA Damage Induces Dynamic Associations of BRD4/P-TEFb With Chromatin and Modulates Gene Transcription in a BRD4-Dependent and -Independent Manner

doi: 10.3389/fmolb.2020.618088

Figure Lengend Snippet: Dynamic recruitments of P-TEFb, Ser2-CTD, and Ser5-CTD of RNAPII onto gene promoter and gene body of BRD4 negatively regulated gene WFIKKNI after UV stress. (A) Representative genomic loci showing BRD4 binding at WFIKKN1 locus. (B) RT-PCR analysis of UV-downregulated gene WFIKKN1 expression after BRD4 knockdown. (C) ChIP-qPCR analysis of isolated DNA associated with CDK9 at both promoter and gene body of WFIKKNI in control and BRD4-depleted HeLa cells after UV treatment (mean ± SD of n = 3). (D) ChIP-qPCR analysis of isolated DNA associated with Ser5-CTD of RNAPII at both promoter and gene body of WFIKKNI in control and BRD4-depleted HeLa cells after UV treatment (mean ± SD of n = 3). (E) ChIP-qPCR analysis of isolated DNA associated with Ser2-CTD of RNAPII at both promoter and gene body of WFIKKNI in control and BRD4-depleted HeLa cells after UV treatment (mean ± SD of n = 3). The data in (B) to (E) are reported as mean values ± SD with the indicated significance by using Student’s t test (* p < 0.05, ** p < 0.01).

Article Snippet: The following antibodies were used in this study: polyclonal anti-rabbit against BRD4 (ab75898), polyclonal anti-rabbit against RNA polymerase II (Santa Cruz Biotechnologies, sc-9001), polyclonal anti-rabbit against RNA polymerase II CTD repeat YSPTSPS (phospho S5) (ab5131), polyclonal anti-rabbit against RNA polymerase II CTD repeat YSPTSPS (phospho S2) (ab5095), polyclonal anti-rabbit against CDK9 (Santa Cruz Biotechnologies, sc-8338), polyclonal anti-rabbit against Cyclin T1 (Santa Cruz Biotechnologies, sc-10750), polyclonal anti-rabbit against HEXIM1 (ab25388), polyclonal anti-rabbit against Histone H3 (ab1791), and monoclonal anti-mouse against TFIIB (ab819).

Techniques: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Knockdown, ChIP-qPCR, Isolation, Control