Journal: Cell Death & Disease

Article Title: ASF1B promotes cervical cancer progression through stabilization of CDK9

doi: 10.1038/s41419-020-02872-5

Figure Lengend Snippet: a Western blots were performed to confirm the IP efficiency. b SDS-PAGE was conducted to analyze the production of IP. c Levels of CDK9 mRNA expression in stable ASF1B-shRNA HeLa cells. d. Protein expression of CDK9 in stable ASF1B-shRNA HeLa cells. e. Co-IP was performed to detect CDK9-ASF1B using anti-ASF1B beads and anti-CDK9 beads. f ASF1B-shRNA-HeLa cells or scrambled cells were treated with CHX at 20 μg/ml for 0, 4, 8, 12, and 16 h, and then the CDK9 expression level was determined by western blot. g ASF1B mediates stabilization through the ubiquitin proteasome pathway. ASF1B-shRNA HeLa cells or control cells were treated with proteasome inhibitor MG132 at 5 μM for 4 h, and then the CDK9 expression level was examined by western blot. h Quantification of the results in ( d ). Data are mean ± SEM, n = 3, and two-tailed unpaired Student’s t test was used. *** p < 0.001. i Colocalization of ASF1B and CDK9 in the nucleus. Immunofluorescent staining and imaging were used to visualize the colocalization of ASF1B (green fluorescence) and CDK9 (red fluorescence) in stable ASF1B-shRNA HeLa cells and corresponding scrambled cells. j A schematic model of this work. A schematic diagram showing the signaling pathway of the ASF1B-mediated effect on cervical cancer cell growth via the ASF1B/CDK9 axis.

Article Snippet: The cells were then incubated with primary antibodies (1:100), including rabbit anti-human ASF1B mAb (Cell Signaling, Danvers, MA) and mouse anti-human CDK9 mAb (Novus Biologicals, Colorado, USA) at 4 °C overnight.

Techniques: Western Blot, SDS Page, Expressing, shRNA, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Control, Two Tailed Test, Staining, Imaging, Fluorescence

Journal: Cell Death & Disease

Article Title: ASF1B promotes cervical cancer progression through stabilization of CDK9

doi: 10.1038/s41419-020-02872-5

Figure Lengend Snippet: The results of proteome analysis.

Article Snippet: The cells were then incubated with primary antibodies (1:100), including rabbit anti-human ASF1B mAb (Cell Signaling, Danvers, MA) and mouse anti-human CDK9 mAb (Novus Biologicals, Colorado, USA) at 4 °C overnight.

Techniques: Functional 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: 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: 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: Nature communications

Article Title: Phosphoproteome profiling uncovers a key role for CDKs in TNF signaling.

doi: 10.1038/s41467-021-26289-6

Figure Lengend Snippet: Fig. 4 TNF-induced cell death leads to a strong RNA-processing response and regulation of the phosphorylation status of CDKs. a Experimental scheme of U937 cells that were untreated, treated with TNF (100 ng/ml, red) alone, treated with TNF and Smac mimetic (SM/Birinapant, 1.25 μM, green) to induce apoptosis, or with TNF, SM, and IDN-6556 (Emricasan, 10 μM, blue) to induce necroptosis. b Z-scored phosphosite intensities of CYLD and XIAP that are regulated upon TNF treatment, TNF-induced apoptosis, and TNF-induced necroptosis (±SEM, n = 4 biologically independent experiments). XIAP phosphosites were retrieved from a DDA dataset (Methods). c Heatmap of z-scored phosphosite intensities that are one-way ANOVA significantly regulated upon TNF treatment or TNF-induced cell death (FDR < 0.05). The profiles are color-coded according to their distance from the respective cluster center (red is close to the center, blue is further away from the center). d Fisher’s exact test on kinases with significantly regulated phosphosites upon different treatments (FDR < 0.02). e Heatmap of means of z-scored phosphosite intensities of CDKs that significantly changed upon treatment of U937 cells (one-way ANOVA, FDR < 0.05). f Heatmap of means of z-scored phosphosite intensities of CDKs that changed significantly (one-way ANOVA) upon treatment of BMDMs with TNF (red) alone, TNF, and TAK1 inhibitor (TAKi, 1 μM) to induce apoptosis (green) or TNF, TAK1i, and the caspase inhibitor IDN-6556 to induce necroptosis (blue). Untreated cells (UT) served as controls (FDR < 0.05). g–i Immunoblots of U937 cells stimulated with TNF as indicated or in combination with CDK inhibitors Dinaciclib (6 nM) (g), THZ531 (200 nM) (h), and AZD4573 (6 nM) (i) and probed with antibodies against phosphorylated (S2) and total (N-terminal) RPB1, phosphorylated (T186), and total CDK9, CDK12, and β-actin (n = 2 biologically independent experiments). Source data are provided as source file.

Article Snippet: Antibodies used for immunoblotting were as follows (diluted 1:1000): anti-human caspase-8 (MBL, M058-3), anti-cleaved human caspase-3 (Cell Signaling Technology CST, 9661), anti-human MLKL (Merck Millipore, MABC604), phospho anti-human RPB1 S2 (Millipore, 04-1571), anti-human RPB1 (CST, D8L4Y), phospho anti-human p65 (CST, 3033 P), anti-human IκBα (CST, 9242), phospho anti-human p38 (CST, 9215), anti-human p38 (CST, 9212), anti-human CDK12 (CST, 11973), phospho anti-human CDK9 (CST, 2549), anti-human CDK9 (CST, 2316), anti-human A20 (Santa Cruz, sc-166692), anti-human MCL1 (CST,4572), anti-human XIAP (MBL, M044-3), anti-human FLIP (CST, 56343) and antihuman β-actin (CST, 4967).

Techniques: Phospho-proteomics, Western Blot

Journal: Nature communications

Article Title: Phosphoproteome profiling uncovers a key role for CDKs in TNF signaling.

doi: 10.1038/s41467-021-26289-6

Figure Lengend Snippet: Fig. 5 CDK9 and CDK12/13 inhibitors potently reduce transcription of TNF-target genes. a Heatmap of z-scored protein intensities significantly changed upon TNF treatment for 6 h in U937 cells (FDR < 0.05). Cells were additionally treated with Dinaciclib (6 nM), CDK12-IN3 (60 nM), and THZ531 (400 nM). Fisher’s exact test on up- and downregulated protein clusters (two-sided Student’s t-test, FDR < 0.001). The profiles are color-coded according to their distance from the respective cluster center (red is close to the center, blue is further away from the center). b Z-scored protein levels of selected TNF-target genes and members of the TNF-signaling pathway (±SD, n = 4 biologically independent experiments). c Immunoblot of U937 cells treated with TNF alone and in combination with the pan-CDK inhibitor Dinaciclib. Proteins were blotted for IκBα, phosphorylated p65, phosphorylated, and total p38 and β-actin (loading control) (n = 2 biologically independent experiments). d qPCR of CCL2 of U937 cells treated for 4 h with TNF alone or in combination with CDK inhibitors Dinaciclib (6 nM), CDK12-IN3 (60 nM), SR4835 (160 nM), THZ531 (200 nM) and AZD4573 (6 nM) (±SD, n = 3 biologically independent experiments). e–h U937 cells treated with TNF alone and in combination with CDK inhibitors (concentrations as in d) were blotted for cFLIP and β-actin (n = 2 biologically independent experiments). Source data are provided as source file.

Article Snippet: Antibodies used for immunoblotting were as follows (diluted 1:1000): anti-human caspase-8 (MBL, M058-3), anti-cleaved human caspase-3 (Cell Signaling Technology CST, 9661), anti-human MLKL (Merck Millipore, MABC604), phospho anti-human RPB1 S2 (Millipore, 04-1571), anti-human RPB1 (CST, D8L4Y), phospho anti-human p65 (CST, 3033 P), anti-human IκBα (CST, 9242), phospho anti-human p38 (CST, 9215), anti-human p38 (CST, 9212), anti-human CDK12 (CST, 11973), phospho anti-human CDK9 (CST, 2549), anti-human CDK9 (CST, 2316), anti-human A20 (Santa Cruz, sc-166692), anti-human MCL1 (CST,4572), anti-human XIAP (MBL, M044-3), anti-human FLIP (CST, 56343) and antihuman β-actin (CST, 4967).

Techniques: Western Blot, Control

Journal: Molecular Cancer Therapeutics

Article Title: Novel Inhibitors of Cyclin-Dependent Kinases Combat Hepatocellular Carcinoma without Inducing Chemoresistance

doi: 10.1158/1535-7163.mct-13-0263

Figure Lengend Snippet: Figure 1. BA-12 and BP-14 diminish cell viability of hepatoma cells and block multiple CDKs. A and B, dose-dependent effects of BA-12 and BP-14 on the viability of human HepG2 (A) and PLC cells (B). C and D, inhibition of CDK1 and CDK2 activity by BA-12 (C) and BP-14 (D) in cell- free extracts. Roscovitine (R) was used at a concentration of 0.03 mmol/L. E and F, reduction of the CDK2-specific phosphorylation of Rb as well as suppression of CDK7 and CDK9 activity after exposure to different concentrations of BA-12 (E) and BP-14 (F) for 24 hours in HepG2 and PLC cells. As detected by immunoblotting, CDK7 and CDK9 activities correspond to serine 5 and serine 2 phosphorylation of RNA polymerase (Pol) II, respectively. The expression of actin indicates equal loading of protein samples. c, control. Error bars depict SD from at least three individual experiments.

Article Snippet: Primary antibodies: anti-S807/811 Rb (Cell Signaling Technology), 1:500; anti-Rb (Cell Signaling Technology), 1:1,000, anti-phospho-Ser5 RNA Pol II (CDK7; Bethyl Laboratories), 1:1,000; anti-phospho-Ser2 RNA Pol II (CDK9; Bethyl Laboratories), 1:1,000; anti-RNA Pol II (Santa Cruz Biotechnology), 1:1,000; anti-PARP (Cell Signaling Technology), 1:1,000; anti-cleaved caspase-7 (Cell Signaling Technology), 1:1,000; anti-caspase-7 (Cell Signaling Technology), 1:1,000; anti-b-actin (Sigma), 1:2,500.

Techniques: Blocking Assay, Inhibition, Activity Assay, Concentration Assay, Phospho-proteomics, Western Blot, Expressing, Control