Journal: bioRxiv

Article Title: Histone chaperone HIRA facilitates transcription elongation to regulate insulin sensitivity and obesity-associated adipose expansion

doi: 10.1101/2025.03.21.644577

Figure Lengend Snippet: 3T3-L1 white preadipocytes were infected with a lentiviral vector expressing human HIRA with C-terminal dTAG and HA double tags, followed by lentiviral CRISPR/Cas9- Hira gRNA to delete endogenous Hira . At D4 of adipogenesis, cells were treated with dTAG-13 for 6h. a , Pie charts depicting the genomic distribution of HIRA binding regions at D4. b , Heat maps were aligned around the center of HIRA binding sites on promoters (6,838), primed enhancers (1,998), active enhancers (AEs, 17,862) and other regions (1,751) at D4. c , Average binding profiles of HIRA-HA, H3K27ac, S5P-Pol II, S2P-Pol II, CDK9 and SPT6 around the center of HIRA + promoters and AEs. Normalized read counts are shown. d , Profiles of HIRA-HA, H3K27ac, S5P-Pol II, S2P-Pol II, CDK9 and SPT6 on gene bodies of HIRA + promoter-associated genes. g , ChIP–Seq profiles of HIRA-HA, H3K27ac, S5P-Pol II, S2P-Pol II, CDK9 and SPT6 were displayed on Adipoq , Mlxipl (ChREBP) and Fasn loci.

Article Snippet: Anti-S5P-Pol II (13523), anti-S2P-Pol II (13499S), anti-CDK9 (2316T) and anti-SPT6 (15616) were from Cell Signaling Technology.

Techniques: Infection, Plasmid Preparation, Expressing, CRISPR, Binding Assay, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: Patient mutation in AIRE disrupts P-TEFb binding and target gene transcription

doi: 10.1093/nar/gkr527

Figure Lengend Snippet: Mutant AIREfsx protein does not interact with P-TEFb. ( A ) Mutant AIREfsx protein does not interact with CDK9. GAL, GAL.AIRE and GAL.AIREfsx chimeras were expressed in HeLa cells. Proteins were immunoprecipitated with anti-GAL antibodies (αGAL) and the co-immunoprecipitation with CDK9 was detected with anti-CDK9 antibodies by western blotting (Top panel, IP:WB). The lower three panels contain 5% of input proteins for immunoprecipitations (WB). ( B ) Mutant AIREfsx protein cannot recruit CDK9 to DNA. Sonicated lysates from HeLa cells co-expressing GAL.AIRE or GAL.AIREfsx fusion proteins and G5HIV2SVEDA were immunoprecipitated with anti-CDK9 (αCDK9) antibodies for ChIP. Enrichment of amplicons is presented as fold enrichment over IgG control (CDK9/IgG). Black and striped bars represent values for the WT GAL.AIRE and mutant GAL.AIREfsx chimeras with SEM depicted by error bars. G5HIV2SVEDA and ChIP amplicons are depicted above and below the bar graph, respectively (promoter, Pr; 5′-end of gene, 5′; 3′-end of gene, 3′, polyadenylation signal, pA; untranscribed region, U). G5HIV2SVEDA contains five GAL binding sites (UAS) and the TATA box (T) upstream of the α-globin gene (αgl) with the inserted fragment from the fibronectin 1 gene (FN1).

Article Snippet: Antibodies used for western blotting, co-immunoprecipitation (co-IP), ChIP or immunofluorescence: anti-AIRE (sc-33188 Santa Cruz Biotechnology), anti-Flag (M2 Sigma-Aldrich), anti-CDK9 (sc-484 Santa Cruz Biotechnology, ab10874 Abcam), anti-RNAPII (ab5408 Abcam, MMS-126 R Covance), anti-S2P (ab5095 Abcam), anti-GAL (sc-510 Santa Cruz Biotechnology), anti-GAPDH (AM4300 Ambion), anti-α Tubulin (DM1A Sigma-Aldrich), anti-U5-116 kD (A300-957A Bethyl labs) rabbit and mouse control IgG (Santa Cruz Biotechnology).

Techniques: Mutagenesis, Immunoprecipitation, Western Blot, Sonication, Expressing, Binding Assay

Journal: Nucleic Acids Research

Article Title: Patient mutation in AIRE disrupts P-TEFb binding and target gene transcription

doi: 10.1093/nar/gkr527

Figure Lengend Snippet: AIRE requires CDK9 for its effects on pre-mRNA splicing. ( A ) Time course of CDK9 siRNA knockdown. HeLa cells were transfected with CDK9 or control siRNAs (cntrl). Subsequently, cells were harvested at various time-points and protein levels were determined with specific antibodies by western blotting. In the upper and lower panels, levels of CDK9 and GAPDH are presented, respectively. ( B ) Protein effectors are expressed in CDK9-knockdown cells. HeLa cells were co-transfected with CDK9 or control siRNAs. GAL or the WT GAL.AIRE chimera and G5HIV2dsx were co-expressed as indicated. Protein levels of expressed proteins are presented in the upper two panels with those of CDK9 and tubulin in the lowest panels, respectively. ( C ) CDK9 siRNA knock-down inhibits effects of AIRE on splicing. Upper panel contains a schematic representation of the G5HIV2dsx minigene with amplicons used to amplify spliced, unspliced or total dsx transcripts by RT–qPCR. Relative splicing efficiency was determined and is presented for the WT GAL.AIRE chimera (black bars) as fold activation relative to GAL (white bars) for cells transfected with CDK9 or control siRNAs. Error bars represent SEM.

Article Snippet: Antibodies used for western blotting, co-immunoprecipitation (co-IP), ChIP or immunofluorescence: anti-AIRE (sc-33188 Santa Cruz Biotechnology), anti-Flag (M2 Sigma-Aldrich), anti-CDK9 (sc-484 Santa Cruz Biotechnology, ab10874 Abcam), anti-RNAPII (ab5408 Abcam, MMS-126 R Covance), anti-S2P (ab5095 Abcam), anti-GAL (sc-510 Santa Cruz Biotechnology), anti-GAPDH (AM4300 Ambion), anti-α Tubulin (DM1A Sigma-Aldrich), anti-U5-116 kD (A300-957A Bethyl labs) rabbit and mouse control IgG (Santa Cruz Biotechnology).

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Activation Assay

Journal: Nucleic Acids Research

Article Title: Patient mutation in AIRE disrupts P-TEFb binding and target gene transcription

doi: 10.1093/nar/gkr527

Figure Lengend Snippet: By recruiting P-TEFb, AIRE induces splicing of an endogenous TRA gene. ( A ) Schematic representation of the KRT14 gene with primer pairs used to amplify spliced and unspliced transcripts by RT–qPCR. ( B ) WT AIRE and mutant AIREfsx proteins are expressed in 293T cells. Expression of proteins was determined with anti-AIRE and anti-α-tubulin antibodies by western blotting (WB). ( C ) Only the WT AIRE protein induces splicing of the KRT14 pre-mRNA. Relative splicing efficiency was determined and is presented for WT AIRE (black bar) and mutant AIREfsx (hashed bar) proteins as fold activation relative to the empty plasmid vector (C, white bar). Error bars represent SEM. ( D ) WT AIRE protein is expressed in 293T cells treated with FP. Expression of proteins was determined with anti-AIRE and anti-α-tubulin antibodies by western blotting (WB). ( E ) FP blocks AIRE-induced enhancement of KRT14 pre-mRNA splicing. Relative splicing efficiency was determined and is presented for the WT AIRE protein (black bars) as fold activation relative to the empty plasmid vector (C, white bars) for treated and untreated cells (FP, DMSO). Error bars represent SEM. ( F ) P-TEFb is enriched on the KRT14 gene in cells expressing the WT AIRE protein. ChIPs were performed with anti-CDK9 (αCDK9) and control IgG antibodies with lysates from 293T cells expressing the empty plasmid vector (C), WT AIRE or mutant AIREfsx proteins. Enrichment of specific KRT14 amplicons is presented as fold enrichment over IgG control (CDK9/IgG). White, black and striped bars represent values for the empty plasmid vector (C), WT AIRE and mutant AIREfsx proteins, respectively, with SEM depicted by error bars. The KRT14 gene and ChIP amplicons are depicted above the bar graph (promoter, Pr; 5′ of coding region, 5′; 3′ of coding region, 3′; polyadenylation signal, pA). ( G ) ChIP with anti-S2P (αS2P) antibodies. Expression levels for AIRE and α-tubulin proteins are presented below the bar graph (WB). Lysates from transfected cells were analyzed with specific antibodies by western blotting.

Article Snippet: Antibodies used for western blotting, co-immunoprecipitation (co-IP), ChIP or immunofluorescence: anti-AIRE (sc-33188 Santa Cruz Biotechnology), anti-Flag (M2 Sigma-Aldrich), anti-CDK9 (sc-484 Santa Cruz Biotechnology, ab10874 Abcam), anti-RNAPII (ab5408 Abcam, MMS-126 R Covance), anti-S2P (ab5095 Abcam), anti-GAL (sc-510 Santa Cruz Biotechnology), anti-GAPDH (AM4300 Ambion), anti-α Tubulin (DM1A Sigma-Aldrich), anti-U5-116 kD (A300-957A Bethyl labs) rabbit and mouse control IgG (Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Mutagenesis, Expressing, Western Blot, Activation Assay, Plasmid Preparation, Transfection

Journal: Genes & Diseases

Article Title: Discovery of a tetrahydroisoquinoline-based CDK9-cyclin T1 protein–protein interaction inhibitor as an anti-proliferative and anti-migration agent against triple-negative breast cancer cells

doi: 10.1016/j.gendis.2021.06.005

Figure Lengend Snippet: ZINC number and docking scores of compounds tested in this study.

Article Snippet: Lipo3000 reagent, control scrambled siRNA (SC-35847, Santa Cruz Biotechnologies, Dallas, TX, USA), CDK9 siRNA 5′-GGAGAAUUUUACUGUGUUUtt-3′ were mixed with DMEM medium for 20 min at 37°C, before adding to cells.

Techniques: Molecular Weight

Journal: Genes & Diseases

Article Title: Discovery of a tetrahydroisoquinoline-based CDK9-cyclin T1 protein–protein interaction inhibitor as an anti-proliferative and anti-migration agent against triple-negative breast cancer cells

doi: 10.1016/j.gendis.2021.06.005

Figure Lengend Snippet: Compound 1 was identified as the top CDK9-cyclin T1 inhibitor. (A) The effect of compounds 1 – 14 on the CDK9-cyclin T1 activity was determined by a chemiluminescence assay. (B – E) Compounds 1 , 2 , 3 , and 6 inhibited CDK9-cyclin T1 activity in a dose-dependent manner as measured using a chemiluminescence assay. (F) The effect of candidate compounds 1 , 2 , 3 , 6 , and 15 on Twist1 expression as detected by Western blotting.

Article Snippet: Lipo3000 reagent, control scrambled siRNA (SC-35847, Santa Cruz Biotechnologies, Dallas, TX, USA), CDK9 siRNA 5′-GGAGAAUUUUACUGUGUUUtt-3′ were mixed with DMEM medium for 20 min at 37°C, before adding to cells.

Techniques: Activity Assay, Chemiluminescence Immunoassay, Expressing, Western Blot

Journal: Genes & Diseases

Article Title: Discovery of a tetrahydroisoquinoline-based CDK9-cyclin T1 protein–protein interaction inhibitor as an anti-proliferative and anti-migration agent against triple-negative breast cancer cells

doi: 10.1016/j.gendis.2021.06.005

Figure Lengend Snippet: Low-energy binding conformation of (A) compound 1 (PDB: 6GZH) (B) ATP (PDB: 3BLQ) and (C) dinaciclib (15) (PDB: 6GZH) generated by in silico docking. (A – C) CDK9 is displayed in ribbon form. Compound 1 (A), ATP (B), and dinaciclib (C) are depicted as a ball-and-stick models showing carbon (yellow), hydrogen (grey), oxygen (red), and nitrogen (blue) atoms. H-bonds are indicated as blue lines. The binding pocket of the CDK9 is represented as a translucent surface. (D) Chemical structure of compound 1 . (E) Lineweaver–Burk plot of enzyme activity measured at different ATP concentrations in the presence of different concentrations of compound 1 . (F) Secondary Lineweaver–Burk plot to obtain the K i value.

Article Snippet: Lipo3000 reagent, control scrambled siRNA (SC-35847, Santa Cruz Biotechnologies, Dallas, TX, USA), CDK9 siRNA 5′-GGAGAAUUUUACUGUGUUUtt-3′ were mixed with DMEM medium for 20 min at 37°C, before adding to cells.

Techniques: Binding Assay, Generated, In Silico, Activity Assay

Journal: Genes & Diseases

Article Title: Discovery of a tetrahydroisoquinoline-based CDK9-cyclin T1 protein–protein interaction inhibitor as an anti-proliferative and anti-migration agent against triple-negative breast cancer cells

doi: 10.1016/j.gendis.2021.06.005

Figure Lengend Snippet: Compound 1 can selectively bind with CDK9 and by block the CDK9-cyclin T1 interaction in in cellulo . (A) MDA-MB-231 cell lysates were treated with compound 1 at 10.0 μM CDK9, Cyclin T1, and β -actin content in the soluble fraction were detected by Western blotting. (B – D) Densitometry analysis of CDK9, cyclin T1, and β -actin content. (E) MDA-MB-231 cell lysates were treated with compound 1 at 10.0 μM CDK1, CDK2, CDK5, CDK9, and CDK12 content in the soluble fraction were detected by Western blotting. (F – J) Densitometry analysis of CDK content. (K) Effect of compound 1 on CDK9-Cyclin T1 interaction as revealed by co-IP assay. Data are represented as mean ± SD. ∗ P < 0.05 vs . DMSO group, (Student's t test).

Article Snippet: Lipo3000 reagent, control scrambled siRNA (SC-35847, Santa Cruz Biotechnologies, Dallas, TX, USA), CDK9 siRNA 5′-GGAGAAUUUUACUGUGUUUtt-3′ were mixed with DMEM medium for 20 min at 37°C, before adding to cells.

Techniques: Blocking Assay, Western Blot, Co-Immunoprecipitation Assay

Journal: Genes & Diseases

Article Title: Discovery of a tetrahydroisoquinoline-based CDK9-cyclin T1 protein–protein interaction inhibitor as an anti-proliferative and anti-migration agent against triple-negative breast cancer cells

doi: 10.1016/j.gendis.2021.06.005

Figure Lengend Snippet: Compound 1 inhibited Snail , Twist1 , and OCT4 at both transcriptional and translational levels. (A – C) Compound 1 inhibited CDK9 at promoters of Snail , Twist1 , and OCT4 gene in MDA-MB-231 cells. ChIP assay was conducted with the primary antibody against CDK9 and IgG. (D – F) Transcriptional levels of snail , Twist1 , and OCT4 in MDA-MB-231 and MDA-MB-468 cells treated with compound 1 or 15 were measured by qPCR. (G – K) Compound 1 inhibited the MDA-MB-231 and MDA-MB-468 stemness by targeting CDK9. Data are represented as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01 vs. DMSO group, (Student's t test).

Article Snippet: Lipo3000 reagent, control scrambled siRNA (SC-35847, Santa Cruz Biotechnologies, Dallas, TX, USA), CDK9 siRNA 5′-GGAGAAUUUUACUGUGUUUtt-3′ were mixed with DMEM medium for 20 min at 37°C, before adding to cells.

Techniques:

Journal: Genes & Diseases

Article Title: Discovery of a tetrahydroisoquinoline-based CDK9-cyclin T1 protein–protein interaction inhibitor as an anti-proliferative and anti-migration agent against triple-negative breast cancer cells

doi: 10.1016/j.gendis.2021.06.005

Figure Lengend Snippet: Compound 1 directly abrogated CDK9 activity via disrupting the CDK9-cyclin T1 PPI and negatively regulating its downstream genes. (A) CDK9 siRNA treatment produced efficient target knockdown in MDA-MB-231 cells. CDK9, cyclin T1, Twist1, Snail, OCT4, CD44, CD133, and β -actin were blotted to control for total protein levels. SiControl: SiRNA control; SiCDK9: SiRNA CDK9. (B–F) Quantification analysis of CDK9, Twist1, Snail, OCT4, CD44, and CD133 in Western blot. Data are represented as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01 vs. DMSO group, (Student's t test).

Article Snippet: Lipo3000 reagent, control scrambled siRNA (SC-35847, Santa Cruz Biotechnologies, Dallas, TX, USA), CDK9 siRNA 5′-GGAGAAUUUUACUGUGUUUtt-3′ were mixed with DMEM medium for 20 min at 37°C, before adding to cells.

Techniques: Activity Assay, Produced, Knockdown, Control, Western Blot

Journal: Genes & Diseases

Article Title: Discovery of a tetrahydroisoquinoline-based CDK9-cyclin T1 protein–protein interaction inhibitor as an anti-proliferative and anti-migration agent against triple-negative breast cancer cells

doi: 10.1016/j.gendis.2021.06.005

Figure Lengend Snippet: Compound 1 inhibited TNBC stemness by targeting CDK9. (A, C) Compound 1 reduced 3D tumor sphere formation in MDA-MB-231 and MDA-MB-468 cells. Scale bars represent 100 μm. (B, D) Relative tumor sphere volume. (E) Effect of compound 1 or 15 (5 μM, 12 h) on CD44 + /CD24 – populations by flow cytometric analysis. Data are represented as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001 vs. DMSO group, (Student's t test).

Article Snippet: Lipo3000 reagent, control scrambled siRNA (SC-35847, Santa Cruz Biotechnologies, Dallas, TX, USA), CDK9 siRNA 5′-GGAGAAUUUUACUGUGUUUtt-3′ were mixed with DMEM medium for 20 min at 37°C, before adding to cells.

Techniques:

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 1| Smad3, Smad4, and cyclin-dependent kinase 9 (CDK9) complex formation in the development and progression of unilateral ureteral obstruction (UUO). (a) Western blotting (WB) demonstrated expression levels of CDK9 in the kidneys 6 h, 12 h, 2 days (d), 4d, and 7d after sham or UUO surgery. (b) Quantitation of relative signal intensities of CDK9/α-tubulin. Data are mean ± s.d., n = 6. *Po0.05, versus sham or UUO 6-h group; #Po0.05, versus UUO 12-h group; $Po0.05, versus UUO 24-h group. (c–l) Confocal microscopy demonstrated the expression of CDK9 (green, d, h), α-smooth muscle actin (α-SMA, red, e, i), merged (f, j, k–m) and DAPI (4,6-diamidino-2-phenylindole; blue, c, g) in the mouse kidney with sham operation (c–f) and kidney with UUO (g–m). c–j, × 600. (k) Area in (j), × 1800. Arrows: samples of CDK9+/α-SMA+ myofibroblasts cells. (l) Quantitation of the percentages of CDK9+/α-SMA+ cells in total DAPI(+) cells. (m) Immunoprecipitation (IP)/WB demonstrated the interactions between Smad3 and Smad4, and Smad3 and CDK9 in the kidneys 6 h, 12 h, 2d, 4d, and 7d after sham or UUO surgery. (n) Quantitation of relative signal intensities of Smad4/Smad3 and CDK9/Smad3. Data are mean ± s.d., n = 6. *Po0.05, versus sham or UUO 6-h group.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Western Blot, Expressing, Quantitation Assay, Confocal Microscopy, Immunoprecipitation

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 3| Smad3, Smad4, and cyclin-dependent kinase 9 (CDK9) complex formation in unilateral ureteral obstruction (UUO). (a) Immunoprecipitation (IP)/western blotting (WB) demonstrated the interactions between CDK9 and Smad3 and CDK9 and Smad4 in the kidneys 7 days after sham or UUO surgery. Quantitation of relative signal intensities of (b) Smad3/CDK9 and (c) Smad4/CDK9 7 days after sham or UUO surgery. Data are mean ± s.d., n = 6. *Po0.05 versus WT sham or WT UUO. NS, P40.05 versus WT UUO. (d) IP/WB demonstrated interactions between Smad4 and CDK9 in the kidneys 7 days after sham or UUO surgery in Smad3 wild-type (WT) or Smad3 knockout (KO) mice. (e) WB demonstrated the expression levels of α-smooth muscle actin (α-SMA), fibronectin (FN), collagen I (Col. I) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase) in the kidneys 7 days after sham or UUO surgery in Smad3 WT or Smad3 KO mice. (f) Quantitation of relative signal intensities of α-SMA, FN, and Col. I in the kidneys 7 days after sham or UUO surgery in Smad3 WT or Smad3 KO mice. Data are mean ± s.d., n = 6. *Po0.05; **Po0.01. (g) IP/WB demonstrated interaction between Smad3 and CDK9, phosphorylated Smad3 T179 (p-T179), and phosphorylated Smad3 C-terminus (p-Tail) in the kidneys 7 days after sham or UUO surgery in Smad4 WT or Smad4 KO. (h) WB demonstrated the expression levels of α-SMA, FN, Col. I, and GAPDH in the kidneys 7 days after sham or UUO surgery in Smad4 WT or Smad4 KO mice. (i) Quantitation of relative signal intensities of α-SMA, FN, and Col. I in the kidneys 7 days after sham or UUO surgery in Smad4 WT or Smad4 KO mice. Data are mean ± s.d., n = 6. *Po0.05; ***Po0.001.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Immunoprecipitation, Western Blot, Quantitation Assay, Knock-Out, Expressing

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 4| Knockdown of cyclin-dependent kinase 9 (CDK9) decreases transforming growth factor-β1 (TGF-β1)-induced Smad3 linker phosphorylation and fibrotic response in renal fibroblasts. (a) Primary cultured mouse renal fibroblasts were transfected with CDK9 siRNA or a scrambled siRNA control. Two days after transfection, renal fibroblasts were stimulated with recombinant TGF-β1 for (a) 2 days or (b) 30 min. (a) Western blotting (WB) shows the expression of CDK9, α-smooth muscle actin (α-SMA), fibronectin, collagen I, and α-tubulin in renal fibroblasts. (b) Immunoprecipitation (IP)/WB or WB shows the interaction between Smad3 and Smad4, Smad3 p-Tail and nuclear p-T179, and the internal control Histone 2A in renal fibroblasts. Quantification of the relative signal intensities of (c) α-SMA/α-tubulin, fibronectin/α-tubulin, collagen I/α-tubulin, (d) Smad4/Smad3, and p-Tail Smad3/Smad3. Data are mean ± s.d., n = 4. *Po0.05;***Po0.001; NS, not significant.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Knockdown, Phospho-proteomics, Cell Culture, Transfection, Control, Recombinant, Western Blot, Expressing, Immunoprecipitation

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 5| Cyclin-dependent kinase 9 (CDK9) promotes transforming growth factor-β1 (TGF-β1)-induced collagen I promoter activity. Western blotting (WB) demonstrated expression levels of (a) nuclear or (b) cytoplasm (Cyto) phosphorylated Smad3 T179 (p-T179) in the kidneys after sham or unilateral ureteral obstruction (UUO) surgery. (c) Immunoprecipitation (IP)/WB demonstrated interactions between CDK9 and Smad3 and CDK9 and Smad4 and the levels of p-T179 after TGF-β1 stimulation in mouse renal fibroblasts. (d) Collagen I promoter luciferase assay demonstrated the effects of CDK9 on Smad3, Smad4 and Smad3, and Smad4 enhancement on collagen I promoter activity with or without TGF-β1 stimulation in mouse renal fibroblasts. Data are mean ± s.d. Experiments were repeated three times. Data were analyzed by two- way analysis of variance for different vector transfections and with or without TGF-β1 treatment. Control versus TGF-β1, Po0.05; a–f represent different levels of collagen I promoter luciferase activity. (e) Collagen I luciferase activity assay demonstrated the effects of CDK9 on Smad3 WT or Smad3 linker-mutated enhancement on collagen I promoter activity in mouse renal fibroblasts. Data are mean ± s.d. Experiments were repeated three times. *Po0.05; **Po0.01. (f–h) Collagen I lunciferase activity assay demonstrated the effects of double knockout of Smad3 and Smad4 (Smad3/4 / ) on CDK9 enhancement (f), kinase-dead CDK9 (dnCDK9) on Smad4 enhancement (g), and 3 serine at Smad3 C-terminus replaced with arginine (3S/A) on CDK9 enhancement (h) on collagen I promoter activity in mouse renal fibroblasts. Data are mean ± s.d. Experiments were repeated three times. *Po0.05; **Po0.01; ***Po0.001.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation, Luciferase, Plasmid Preparation, Transfection, Control, Double Knockout

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 6| Cyclin-dependent kinase 9 (CDK9) inhibitor inhibits transforming growth factor-β1 (TGF-β1)-induced fibrotic response in renal fibroblasts. (a) Collagen I promoter luciferase assay demonstrated effects of CDK9 inhibitor (CDK9i) and Smad3 inhibitor (SIS3) on collagen I promoter activity with or without TGF-β1 stimulation in mouse renal fibroblasts. Data are mean ± s.d. Experiments were repeated three times. Data were analyzed by one-way analysis of variance for different treatments. *Po0.05 versus control; #Po0.05 versus TGF-β1; $Po0.05 versus TGF-β1+CDK9i 50 nM. (b) Western blotting (WB) demonstrated the expression levels of α-smooth muscle actin (α-SMA), collagen I (Col. I), and fibronectin (FN), and internal control α-tubulin 3 days after different treatments in mouse renal fibroblasts. (c) WB demonstrated nuclear phosphorylated Smad3 T179 (Smad3 p-T179) and phosphorylated RNAPII Ser5 (RNAPII p-Ser5) for the indicated period of time of treatments in mouse renal fibroblasts.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Luciferase, Activity Assay, Control, Western Blot, Expressing

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 7| Smad3, Smad4, and cyclin-dependent kinase 9 (CDK9) complex formation in 2-day unilateral ureteral obstruction (UUO) after administration of CDKi or/and SiS3. (a) Western blotting (WB) demonstrated the expression levels of nuclear phosphorylated Smad3 T-179 (p-T179) and Smad3 C-terminus (p-Tail Smad3) in the kidneys 2d after sham or UUO surgery. (b) Quantitation of relative signal intensities of p-T179/Histone 2A and p-Tail Smad3/Smad3. Data are mean ± s.d., n = 6. *Po0.05, versus sham group; #Po0.05, versus UUO vehicle or UUO CDK9i 1 μg/g/day group. (c) Immunoglobulin (IP)/WB demonstrated the interactions between Smad3 and Smad4, Smad3 and CDK9, and Smad4 and CDK9 in the kidneys 2d after sham or UUO surgery. (d) Quantitation of relative signal intensities of Smad4/Smad3, Smad3/CDK9, and Smad4/CDK9. Data are mean ± s.d., n = 6. *Po0.05, versus sham group; #Po0.05, versus UUO vehicle group; $Po0.05, versus UUO CDK9i 4 μg/g/day group or UUO SIS3 2.5 μg/g/day group.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Western Blot, Expressing, Quantitation Assay

Journal: Kidney international

Article Title: The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

doi: 10.1038/ki.2015.235

Figure Lengend Snippet: Figure 8| The effects of cyclin-dependent kinase 9 inhibitor (CDK9i) and SiS3 on renal fibrosis and inflammation in unilateral ureteral obstruction (UUO). (a) Western blotting (WB) demonstrated the expression levels of α-smooth muscle actin (α-SMA), collagen I (Col. I), and fibronectin (FN) and internal control α-tubulin in the kidneys 7d after sham or UUO surgery with different treatments. (b) Quantitation of relative signal intensities of α-SMA/α-tubulin, Col. I/α-tubulin, and FN/α-tubulin. Data are mean ± s.d., n = 6. *Po0.05, versus sham group; #Po0.05, versus UUO vehicle group; $Po0.05, versus UUO CDK9i 4 μg/g/day group or UUO SiS3 2.5 μg/g/day group. (c) Quantitation of the number of F4/80+ macrophages in the kidneys 7d after sham or UUO surgery with different treatments. Data are mean ± s.d., n = 6. *Po0.05, versus sham group; #Po0.05, versus UUO vehicle group; $Po0.05, versus UUO CDK9i 4 μg/g/day group or UUO SiS3 2.5 μg/g/day group.

Article Snippet: After blocking for 30 min at 4 °C in 5% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20, the membrane was incubated overnight with rabbit anti-Smad3, CDK9, p-Smad3 Thr179 (Biorbyt, Cambridge, UK), or rabbit antiphospho-Smad3 (Ser423/425) (Cell signaling Technology), or Smad4 (Cell Signalling Technology).

Techniques: Western Blot, Expressing, Control, Quantitation Assay

Journal: Molecular and Cellular Biology

Article Title: CSB-Dependent Cyclin-Dependent Kinase 9 Degradation and RNA Polymerase II Phosphorylation during Transcription-Coupled Repair

doi: 10.1128/MCB.00225-18

Figure Lengend Snippet: CDK9 function during transcription restart after DNA repair. (A) Western blot showing the expression of CDK9 in MRC5 whole-cell extract after siRNA mediated knockdown of CDK9 in nonirradiated cells and 24h after irradiation. UBF serves as a loading control. (B) Quantification of at least three different experiments of CDK9 level normalized to UBF in nonirradiated cells and siCDK9 compared to siMock. (C and D) RNA synthesis in MRC5 cells after siRNA-mediated knockdown of the indicated factors in nonirradiated cells (C) and after UV-C exposure (D). At least 60 nuclei were analyzed. (E) Western blot showing the expression of CDK9 in MRC5 whole-cell extract after 6 h of treatment with CDK9 inhibitor (flavopiridol or iCDK9). UBF serves as a loading control. (F) RNA synthesis in MRC5 cells after 3 and 24 h of treatment with CDK9 inhibitor. At least 50 nuclei were analyzed. Error bars represent the standard errors of the mean (SEM). P values: *, <0.05; ***, <0.001.

Article Snippet: For CDK9 kinase inhibition, cells were treated with 0.5 µM flavopiridol (Sigma, F3055) or 0.5 µM iCDK9 (MedChemExpress, HY-16462/CS-1252).

Techniques: Western Blot, Expressing, Knockdown, Irradiation, Control