Journal: bioRxiv

Article Title: Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

doi: 10.1101/2025.11.23.690049

Figure Lengend Snippet: ( A ) Chemical structure of Q901. ( B ) Assessment of the covalent binding site was conducted via mass spectrometry analysis of the recombinant CDK7-cyclin H-MAT1 (CAK) trimetric complex. The recombinant CAK complex was incubated with Q901 or DMSO for 1 h, digested for 18 h, and analyzed by mass spectrometry. ( C ) KinMap image illustrating the kinase inhibition profile of Q901 against a panel of 410 kinases (397 protein kinase assays and 13 lipid kinase assays). The inhibition profile was determined by measuring the residual activity at 1 μM for 1 h using the PanQinase® Activity Assay. ATP concentration was set at the apparent ATP-Km value for each kinase. A red dot indicates 99% inhibition of CDK7 by Q901 at this concentration. ( D ) Efficacy and selectivity of Q901 against other CDKs at ATP concentrations corresponding to the apparent ATP-Km value for each kinase. Residual activity (%) was measured after a 1 h incubation with Q901 at the indicated concentrations. ( E ) CDK7 target occupancy assay using Bio-QS, a biotinylated analog of Q901. Cell lysates were prepared from A2780 cells treated with Q901 or DMSO for 4 h at the indicated concentrations and subjected to immunoprecipitation (IP) using Bio-QS and streptavidin agarose beads (SA). IP samples and whole-cell lysates were immunoblotted with an anti-CDK7 antibody. ( F ) Washout-based target occupancy assay to measure the duration of CDK7 inhibition. A2780 cells treated with 6 nM Q901 for 4 h were washed with fresh medium and incubated for the indicated times. Cells were lysed, treated with Bio-QS, and immunoprecipitated with streptavidin agarose beads (SA). The percentage of free CDK7 was calculated by normalizing CDK7 levels in IP samples from Q901 treatment to those in IP samples from the DMSO-treated group (n = 3; two-way ANOVA with Tukey’s multiple comparisons test, data represent mean ± SD).

Article Snippet: The following antibodies were used for immunoblotting: total CDK7 (Cell Signaling Technology, Danvers, MA, USA, 2090S, RRID: AB_2077140), MYC (Cell Signaling Technology, 13987, RRID: AB_2631168), E2F1 (Cell Signaling Technology, 3742, RRID: AB_2096936), MYC S62P (Cell Signaling Technology, 13748, RRID: AB_2687518), MYC T58P (Cell Signaling Technology, 46650), Rb S795P (Cell Signaling Technology, 9301, RRID: AB_330013), Rb S780P (Cell Signaling Technology, 9307, RRID: AB_330015), MAX (Cell Signaling Technology, 4739, RRID: AB_2281777), T-H2AX (Santa Cruz, Dallas, TX, USA, sc-517336, RRID: AB_3675923), P-H2AX (Cell Signaling Technology, 2577, RRID: AB_2118010), Ubiquitin (Santa Cruz, sc-8017, RRID: AB_628423), TOP1 (BD Biosciences, San Jose, CA, USA, 556597, RRID: AB_396474), β-actin (Santa Cruz, sc-47778, RRID: AB_626632), Affinity Purified Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, 1706515, RRID: AB_11125142), and Affinity Purified Goat Anti-Mouse IgG (H+L)-HRP (Bio-Rad, 1706516, RRID: AB_11125547).

Techniques: Binding Assay, Mass Spectrometry, Recombinant, Incubation, Inhibition, Activity Assay, Concentration Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

doi: 10.1101/2025.11.23.690049

Figure Lengend Snippet: A ) 1 H NMR spectrum of Q901 was acquired using variable temperature (VT) NMR in DMSO-d□. ( B ) Targeted proteomics analysis to determine the Q901 binding sites on CDK7. The recombinant CAK trimeric complex was incubated with Q901 or DMSO, followed by protease digestion and peptide mapping via LC-MS/MS. Chromatograms show peptide fragments generated by ArgC (Clostripain) digestion (right) and ArgC/Trypsin digestion (left). The expanded boxes highlight the peak of C312 containing peptides, which are reduced following Q901 treatment, indicating covalent modification at this site. ( C ) Representative Western blot images from the pulse-chase assay described in . A2780 cells were treated with 6 nM Q901 for 4 h and then divided into two groups. One group (- wash out) remained in the Q901-containing medium for continuous incubation, while the other group (+ wash out) underwent a drug washout, where the medium was completely removed and replaced with fresh drug-free medium before further incubation for the indicated times. Bio-QS-labeled CDK7 was immunoprecipitated using streptavidin agarose beads (SA), and the levels of free CDK7 were analyzed by immunoblotting. These images in this figure were quantified in .

Article Snippet: The following antibodies were used for immunoblotting: total CDK7 (Cell Signaling Technology, Danvers, MA, USA, 2090S, RRID: AB_2077140), MYC (Cell Signaling Technology, 13987, RRID: AB_2631168), E2F1 (Cell Signaling Technology, 3742, RRID: AB_2096936), MYC S62P (Cell Signaling Technology, 13748, RRID: AB_2687518), MYC T58P (Cell Signaling Technology, 46650), Rb S795P (Cell Signaling Technology, 9301, RRID: AB_330013), Rb S780P (Cell Signaling Technology, 9307, RRID: AB_330015), MAX (Cell Signaling Technology, 4739, RRID: AB_2281777), T-H2AX (Santa Cruz, Dallas, TX, USA, sc-517336, RRID: AB_3675923), P-H2AX (Cell Signaling Technology, 2577, RRID: AB_2118010), Ubiquitin (Santa Cruz, sc-8017, RRID: AB_628423), TOP1 (BD Biosciences, San Jose, CA, USA, 556597, RRID: AB_396474), β-actin (Santa Cruz, sc-47778, RRID: AB_626632), Affinity Purified Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, 1706515, RRID: AB_11125142), and Affinity Purified Goat Anti-Mouse IgG (H+L)-HRP (Bio-Rad, 1706516, RRID: AB_11125547).

Techniques: Targeted Proteomics, Binding Assay, Recombinant, Incubation, Liquid Chromatography with Mass Spectroscopy, Generated, Modification, Western Blot, Pulse Chase, Labeling, Immunoprecipitation

Journal: bioRxiv

Article Title: Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

doi: 10.1101/2025.11.23.690049

Figure Lengend Snippet: ( A ) MCF-7 cells were treated with Q901 at the indicated concentrations for 72, 96, or 120 h. Cell viability was measured using the ATP Lite™ system. Inhibition (%) was plotted against the log-transformed Q901 concentration (µM) (n = 3 to 4). Data represent mean ± SD. ( B and C ) RNAPII ChIP-seq was performed following treatment with 100 nM Q901 for the indicated duration. (B) Volcano plot of pan RNAPII ChIP-seq signals after Q901 treatment (n = 2; blue dot indicates p-value ≤ 0.05 and log2FC ≤ -0.58; red dot indicates p-value ≤ 0.05 and log2FC ≥ 0.58). (C) Average ChIP-seq signal plots of various RNAPII forms for genes downregulated by Q901. ( D ) Average CDK7 ChIP-seq signal plots of downregulated and upregulated genes at the TSS. CDK7 ChIP-seq was performed with 100 nM Q901 for the indicated durations.

Article Snippet: The following antibodies were used for immunoblotting: total CDK7 (Cell Signaling Technology, Danvers, MA, USA, 2090S, RRID: AB_2077140), MYC (Cell Signaling Technology, 13987, RRID: AB_2631168), E2F1 (Cell Signaling Technology, 3742, RRID: AB_2096936), MYC S62P (Cell Signaling Technology, 13748, RRID: AB_2687518), MYC T58P (Cell Signaling Technology, 46650), Rb S795P (Cell Signaling Technology, 9301, RRID: AB_330013), Rb S780P (Cell Signaling Technology, 9307, RRID: AB_330015), MAX (Cell Signaling Technology, 4739, RRID: AB_2281777), T-H2AX (Santa Cruz, Dallas, TX, USA, sc-517336, RRID: AB_3675923), P-H2AX (Cell Signaling Technology, 2577, RRID: AB_2118010), Ubiquitin (Santa Cruz, sc-8017, RRID: AB_628423), TOP1 (BD Biosciences, San Jose, CA, USA, 556597, RRID: AB_396474), β-actin (Santa Cruz, sc-47778, RRID: AB_626632), Affinity Purified Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, 1706515, RRID: AB_11125142), and Affinity Purified Goat Anti-Mouse IgG (H+L)-HRP (Bio-Rad, 1706516, RRID: AB_11125547).

Techniques: Inhibition, Transformation Assay, Concentration Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

doi: 10.1101/2025.11.23.690049

Figure Lengend Snippet: ( A ) The results of SE calling using the ROSE program with H3K27ac ChIP-seq (GSE62229). ( B ) Expression levels of enhancer target genes (pan RNAPII ChIP-seq; n = 2, Q901 1h treatment condition, data represent mean ± SEM). ( C ) Average fastGRO signals of four enhancer groups. ( D ) GO analysis results of target genes regulated by CDK7-bound SE and CDK7-bound TE. ( E ) Track images showing ChIP-seq signals for H3K27ac, CDK7, pan RNAPII, MYC, and E2F1, along with fastGRO, at a representative CDK7-bound SE region (highlighted in yellow) and it associated target genes.

Article Snippet: The following antibodies were used for immunoblotting: total CDK7 (Cell Signaling Technology, Danvers, MA, USA, 2090S, RRID: AB_2077140), MYC (Cell Signaling Technology, 13987, RRID: AB_2631168), E2F1 (Cell Signaling Technology, 3742, RRID: AB_2096936), MYC S62P (Cell Signaling Technology, 13748, RRID: AB_2687518), MYC T58P (Cell Signaling Technology, 46650), Rb S795P (Cell Signaling Technology, 9301, RRID: AB_330013), Rb S780P (Cell Signaling Technology, 9307, RRID: AB_330015), MAX (Cell Signaling Technology, 4739, RRID: AB_2281777), T-H2AX (Santa Cruz, Dallas, TX, USA, sc-517336, RRID: AB_3675923), P-H2AX (Cell Signaling Technology, 2577, RRID: AB_2118010), Ubiquitin (Santa Cruz, sc-8017, RRID: AB_628423), TOP1 (BD Biosciences, San Jose, CA, USA, 556597, RRID: AB_396474), β-actin (Santa Cruz, sc-47778, RRID: AB_626632), Affinity Purified Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, 1706515, RRID: AB_11125142), and Affinity Purified Goat Anti-Mouse IgG (H+L)-HRP (Bio-Rad, 1706516, RRID: AB_11125547).

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

Article Title: Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

doi: 10.1101/2025.11.23.690049

Figure Lengend Snippet: ( A ) Average ChIP-seq signal plots of CDK7 and pan RNAPII ChIP-seq across four enhancer groups. ( B ) Average ChIP-seq signal plots for CDK7 and pan RNAPII for protein-coding genes regulated by four enhancer groups. ( C ) Bar graph shows the proportion of downregulated genes in each enhancer groups (Pan RNAPII ChIP-seq; n = 2; p-value ≤ 0.05 and log2FC ≤ -0.58). ( D ) Scatter plot shows the expression levels and log2FC of target genes regulated by CDK7-bound SE and CDK7-bound TE (pan RNAPII ChIP-seq; n = 2; p-value ≤ 0.05).

Article Snippet: The following antibodies were used for immunoblotting: total CDK7 (Cell Signaling Technology, Danvers, MA, USA, 2090S, RRID: AB_2077140), MYC (Cell Signaling Technology, 13987, RRID: AB_2631168), E2F1 (Cell Signaling Technology, 3742, RRID: AB_2096936), MYC S62P (Cell Signaling Technology, 13748, RRID: AB_2687518), MYC T58P (Cell Signaling Technology, 46650), Rb S795P (Cell Signaling Technology, 9301, RRID: AB_330013), Rb S780P (Cell Signaling Technology, 9307, RRID: AB_330015), MAX (Cell Signaling Technology, 4739, RRID: AB_2281777), T-H2AX (Santa Cruz, Dallas, TX, USA, sc-517336, RRID: AB_3675923), P-H2AX (Cell Signaling Technology, 2577, RRID: AB_2118010), Ubiquitin (Santa Cruz, sc-8017, RRID: AB_628423), TOP1 (BD Biosciences, San Jose, CA, USA, 556597, RRID: AB_396474), β-actin (Santa Cruz, sc-47778, RRID: AB_626632), Affinity Purified Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, 1706515, RRID: AB_11125142), and Affinity Purified Goat Anti-Mouse IgG (H+L)-HRP (Bio-Rad, 1706516, RRID: AB_11125547).

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

Article Title: Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

doi: 10.1101/2025.11.23.690049

Figure Lengend Snippet: ( A and B ) Average ChIP-seq signal plots of CDK7 (A) and pan RNAPII (B) for gene sets related to DNA repair, MYC targets V1, and E2F targets. ( C ) Track image of SRSF6 gene, a representative gene from the DNA repair pathway. ( D ) Track image of RPLP0 gene, a representative gene from the MYC targets V1 pathway. ( E ) Track image of EZH2 gene, a representative gene from the E2F targets pathway. ( F ) Heatmap showing log2FC values of DNA damage/repair genes expression from pan RNAPII ChIP-seq and mRNA-seq data (right; n = 3, FDR ≤ 0.1).

Article Snippet: The following antibodies were used for immunoblotting: total CDK7 (Cell Signaling Technology, Danvers, MA, USA, 2090S, RRID: AB_2077140), MYC (Cell Signaling Technology, 13987, RRID: AB_2631168), E2F1 (Cell Signaling Technology, 3742, RRID: AB_2096936), MYC S62P (Cell Signaling Technology, 13748, RRID: AB_2687518), MYC T58P (Cell Signaling Technology, 46650), Rb S795P (Cell Signaling Technology, 9301, RRID: AB_330013), Rb S780P (Cell Signaling Technology, 9307, RRID: AB_330015), MAX (Cell Signaling Technology, 4739, RRID: AB_2281777), T-H2AX (Santa Cruz, Dallas, TX, USA, sc-517336, RRID: AB_3675923), P-H2AX (Cell Signaling Technology, 2577, RRID: AB_2118010), Ubiquitin (Santa Cruz, sc-8017, RRID: AB_628423), TOP1 (BD Biosciences, San Jose, CA, USA, 556597, RRID: AB_396474), β-actin (Santa Cruz, sc-47778, RRID: AB_626632), Affinity Purified Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, 1706515, RRID: AB_11125142), and Affinity Purified Goat Anti-Mouse IgG (H+L)-HRP (Bio-Rad, 1706516, RRID: AB_11125547).

Techniques: ChIP-sequencing, Expressing

Journal: bioRxiv

Article Title: Sensitizing tumor response to topoisomerase I antibody drug conjugate by selective CDK7 inhibition

doi: 10.1101/2025.11.23.690049

Figure Lengend Snippet: ( A ) A model illustrating how Q901 enhances the activity of TOP1i and TOP1i-ADCs. (Left) Q901 promotes CDK7 accumulation at the TSS while reducing RNAPII binding. This also decreases MYC and E2F1 binding at the TSS, leading to downregulation of genes involved in the DNA damage response pathway. (Middle) The dual inhibition of CDK7 (by Q901) and TOP1 (by TOP1i) blocks the repair of TOP1i-induced DNA damage, ultimately leading to cell death. (Right) The combination of Q901 and a TOP1i-ADC shows potent enhanced anticancer activity, effectively inducing cancer cell death in vitro and significantly reducing tumor growth in vivo. ( B and C ) HCT116, HER2 ultra low/negative human colon cancer cell line, was treated with Q901, T-DXd (10 μg/ml), or their combination at the indicated concentrations for 72 h (B). Dose-response curves were plotted as a function of log-transformed concentration relative to IC□□ values. Cell viability was measured using the ATP Lite™ system (n = 2, data represent mean ± SD). (C) For in vivo efficacy study, HCT116 cells mixed with Matrigel (1:1) were subcutaneously implanted into BALB/c nude mice. When tumors reached an average size of 117 mm3, mice were randomized into groups (n = 8 per group) and treated with Q901 (10 mg/kg, intraperitoneally once daily), T-DXd alone (10 mg/kg, intravenous single injection on day 0) or the combination. ( D and F ) H292, TROP2 positive human lung cancer cell line, was treated with Q901 in combination with SG at the indicated concentrations for 72 h (D). Dose-response curves were generated using log-transformed concentrations normalized to IC□□ values. Cell viability was assessed using the ATP Lite™ system (n = 2, data represent mean ± SD). ( E and F ) For in vivo efficacy study, H292 cells were mixed with Matrigel (1:1) and implanted subcutaneously into BALB/c nude mice. When tumors reached an average size of 140 mm3, mice were randomized into groups (n = 8 per group) and treated with Q901 (10 or 3 mg/kg, intraperitoneally once daily), SG alone (3 or 10 mg/kg, intravenous single injection on day 1 and 8) or the combination of both. The graph shows the mean tumor volume ± SEM. Statistical significance was calculated using GraphPad Prism software (*: p < 0.01, ****: p < 0.0001 by two-way ANOVA followed by Tukey’s multiple comparison test).

Article Snippet: The following antibodies were used for immunoblotting: total CDK7 (Cell Signaling Technology, Danvers, MA, USA, 2090S, RRID: AB_2077140), MYC (Cell Signaling Technology, 13987, RRID: AB_2631168), E2F1 (Cell Signaling Technology, 3742, RRID: AB_2096936), MYC S62P (Cell Signaling Technology, 13748, RRID: AB_2687518), MYC T58P (Cell Signaling Technology, 46650), Rb S795P (Cell Signaling Technology, 9301, RRID: AB_330013), Rb S780P (Cell Signaling Technology, 9307, RRID: AB_330015), MAX (Cell Signaling Technology, 4739, RRID: AB_2281777), T-H2AX (Santa Cruz, Dallas, TX, USA, sc-517336, RRID: AB_3675923), P-H2AX (Cell Signaling Technology, 2577, RRID: AB_2118010), Ubiquitin (Santa Cruz, sc-8017, RRID: AB_628423), TOP1 (BD Biosciences, San Jose, CA, USA, 556597, RRID: AB_396474), β-actin (Santa Cruz, sc-47778, RRID: AB_626632), Affinity Purified Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad, Hercules, CA, USA, 1706515, RRID: AB_11125142), and Affinity Purified Goat Anti-Mouse IgG (H+L)-HRP (Bio-Rad, 1706516, RRID: AB_11125547).

Techniques: Activity Assay, Binding Assay, Inhibition, In Vitro, In Vivo, Transformation Assay, Concentration Assay, Injection, Generated, Software, Comparison

Journal: Molecular Cancer Therapeutics

Article Title: Triptolide Induces Cell Killing in Multidrug-Resistant Tumor Cells via CDK7/RPB1 Rather than XPB or p44

doi: 10.1158/1535-7163.mct-15-0753

Figure Lengend Snippet: Figure 3. Triptolide induces RPB1 degradation in both parental and MDR cell lines and RPB1 phosphorylation at Ser1878. A, MES/SA, MES-SA/MX5, KB, and KB/VCR cells were treated as shown in Materials and Methods and immunoblotted for RPB1. B, SK-OV-3, KB, and KB/VCR cells were pretreated with a CDK7- specific inhibitor and then assayed for RPB1. C, SK-OV-3 cells were treated as indicated and immunoblotted for phosphorylated RPB1 at Ser5 and phosphorylated CDK7 at Thr170. D, KB and KB/VCR cells were treated as indicated and immunoblotted for phosphorylated RPB1 at Ser5 and phosphorylated CDK7 at Thr170. E, cells were cultured as depicted in Materials and Methods, and IC50 values were measured using a Cell Counting Kit-8 (CCK-8) assay. F, KB and KB/VCR cells were cultured as indicated and immunoblotted for RPB1. G, SK-OV-3 cells were treated as indicated and immunoprecipitated RPB1 and separated with SDS-PAGE. Gel bands were excised, digested, and assessed using LC/MS-MS to identify the phosphorylation site of RPB1. m/z, mass-to-charge ratio.

Article Snippet: All antibodies were commercially available: RPB1 [carboxy-terminal domain (CTD) repeat], p-S5-RPB1, XPB, OCT-4, SOX-2, NANOG, ubiquitin, and p-Thr170-CDK7were fromAbcam,GAPDHand IgGwere from Beyotime Institute of Biotechnology (Haimen, China), CDK7 and p44 were from Santa Cruz Biotechnology, and c-MYC was from BD Biosciences.

Techniques: Phospho-proteomics, Cell Culture, Cell Counting, CCK-8 Assay, Immunoprecipitation, SDS Page, Liquid Chromatography with Mass Spectroscopy

Journal: Virus research

Article Title: Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

doi: 10.1016/j.virusres.2023.199200

Figure Lengend Snippet: Fig. 1. Structural model of the interaction between pUL97 and cyclin H. (A) Predicted binding site of pUL97(231-280) (orange) superimposed with the experimental cyclin H–CDK7–MAT1 complex structure (gray, cyan, and dark blue). This model suggests that pUL97(231-280) uses the same binding pocket as MAT1 for targeting the cyclin H–CDK7 complex. (B) Model of a ternary pUL97–cyclin H–CDK7 complex, in which pUL97 is attached to cyclin H exclusively through IF2 formed by the 231-280 sequence stretch. The pUL97 kinase domain (residues 329-634, marked in red) is connected to the complex by a nonstructured, flexible linker (residues 281-328, indicated as dark orange connecting line). (C) Model of a pUL97–cyclin H complex, in which pUL97 interacts with cyclin H both through IF2, pUL97(231-280) (orange), and the globular kinase domain IF1, pUL97(329-634) (red), thereby displacing CDK7.

Article Snippet: The following antibodies were used in this study: mAb-UL97.01, mAb-UL53, mAb-UL50, and mAb-UL56 (kindly provided by T. Lenac and S. Jonick, University of Rijeka, Croatia), mAb-IE1 (kindly provided by William Britt, Birmingham, AL, USA), mAb-UL44 and mAb-MCP (kindly provided by Bodo Plachter, University of Mainz, Mainz, Germany), mAb-pp65 and mAb-UL69 (kindly provided by T. Stamminger, Ulm, Germany), mAb-β-actin (A5441, Sigma Aldrich), pAb-cyclin H (LSC331195, LS Bio), pAb-CDK7 pSer164 (PA5-105583, Sigma-Aldrich), pAb-CDK7 pThr170 (ab155976, Abcam), pAb-CDK9 pThr186 (2549, Cell signaling), mAb-CDK7 (sc-7344, Santa Cruz Biotechnology), pAbRNAPII CTD pSer2 (NB100-1805, Novus Biologicals), mAb-Flag (F1804, Sigma-Aldrich).

Techniques: Binding Assay, Sequencing

Journal: Virus research

Article Title: Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

doi: 10.1016/j.virusres.2023.199200

Figure Lengend Snippet: Fig. 3. Viral protein substrate phosphorylation by CDK7. HFFs were infected with HCMV AD169-GFP at MOI of 1 and lysed 3 d.p.i. (A) Viral proteins were coimmunoprecipitated with cellular kinase CDK7 (lanes 3-10) and exposed to the non-radioactive IVKA reaction. Specific phosphorylation signals are marked to indicate single (◦) or continuous (arrows in respective colors) bands. Autophosphorylation signals of viral kinase pUL97 served as a positive control (lane 2). Du plicates of each immunoprecipitate were treated with 5 µM of CDK7-inhibitor LDC4297 shortly before the IVKA reaction (lanes 4, 6, 8, 10). (B) An IVKA reaction conducted in parallel without ATPγS served as a negative control. Successful IP (C) and reliable expression levels (D) of all proteins of interest were demonstrated by Wb analysis. (E) Phosphorylation signals from IVKA reactions shown in (A) were quantitated three times with varying background parameters, resulting in triplicate determinations of each sample, and mean values ± SD were then normalized to IP control.

Article Snippet: The following antibodies were used in this study: mAb-UL97.01, mAb-UL53, mAb-UL50, and mAb-UL56 (kindly provided by T. Lenac and S. Jonick, University of Rijeka, Croatia), mAb-IE1 (kindly provided by William Britt, Birmingham, AL, USA), mAb-UL44 and mAb-MCP (kindly provided by Bodo Plachter, University of Mainz, Mainz, Germany), mAb-pp65 and mAb-UL69 (kindly provided by T. Stamminger, Ulm, Germany), mAb-β-actin (A5441, Sigma Aldrich), pAb-cyclin H (LSC331195, LS Bio), pAb-CDK7 pSer164 (PA5-105583, Sigma-Aldrich), pAb-CDK7 pThr170 (ab155976, Abcam), pAb-CDK9 pThr186 (2549, Cell signaling), mAb-CDK7 (sc-7344, Santa Cruz Biotechnology), pAbRNAPII CTD pSer2 (NB100-1805, Novus Biologicals), mAb-Flag (F1804, Sigma-Aldrich).

Techniques: Phospho-proteomics, Infection, Positive Control, Negative Control, Expressing, Control

Journal: Virus research

Article Title: Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

doi: 10.1016/j.virusres.2023.199200

Figure Lengend Snippet: Fig. 6. Human CDK7 does not increase pUL97 in vitro kinase activity. (A) 293T cells were transiently transfected with plasmids encoding pUL97-Flag. Cells were lysed 2 d post-transfection, and pUL97-Flag and CDK7 were immu noprecipitated using the indicated antibodies; a mouse Fc fragment served as a negative control. Dynabeads-bound proteins were eluted in 150 µl enzyme buffer. 25 µl of eluted Dynabeads were denatured in 25 µl 2x loading buffer and subjected to SDS-PAGE and Wb to confirm successful precipitation of pUL97- Flag and CDK7. (B) Samples derived from immunoprecipitation of CDK7 plus pUL97-Flag, as well as Fc control samples, were subjected to a qSox-IVKA (using the pUL97-specific sensor peptide AQT0258). Optionally, 0.01 µM of CDK7-specific inhibitor LDC4297 (corresponds to the mean antiviral EC50 value) or equal amounts of DMSO were added to the reactions. Background activity determined with the Fc control was subtracted from values of combined pUL97-Flag plus CDK7 kinase activity. Mean values ± SD derived from one representative experiment are given, as derived from measurements in tripli cates. Statistical analysis was performed using an ordinary two-way ANOVA: n. s., not significant.

Article Snippet: The following antibodies were used in this study: mAb-UL97.01, mAb-UL53, mAb-UL50, and mAb-UL56 (kindly provided by T. Lenac and S. Jonick, University of Rijeka, Croatia), mAb-IE1 (kindly provided by William Britt, Birmingham, AL, USA), mAb-UL44 and mAb-MCP (kindly provided by Bodo Plachter, University of Mainz, Mainz, Germany), mAb-pp65 and mAb-UL69 (kindly provided by T. Stamminger, Ulm, Germany), mAb-β-actin (A5441, Sigma Aldrich), pAb-cyclin H (LSC331195, LS Bio), pAb-CDK7 pSer164 (PA5-105583, Sigma-Aldrich), pAb-CDK7 pThr170 (ab155976, Abcam), pAb-CDK9 pThr186 (2549, Cell signaling), mAb-CDK7 (sc-7344, Santa Cruz Biotechnology), pAbRNAPII CTD pSer2 (NB100-1805, Novus Biologicals), mAb-Flag (F1804, Sigma-Aldrich).

Techniques: In Vitro, Activity Assay, Transfection, Negative Control, SDS Page, Derivative Assay, Immunoprecipitation, Control

Journal: Virus research

Article Title: Cytomegalovirus cyclin-dependent kinase ortholog vCDK/pUL97 undergoes regulatory interaction with human cyclin H and CDK7 to codetermine viral replication efficiency.

doi: 10.1016/j.virusres.2023.199200

Figure Lengend Snippet: Fig. 7. Phosphorylation patterns of CDK7 depend on the state of HCMV infection and pUL97 kinase activity. (A) HFFs were seeded in T75 cell culture flasks and infected 1 d later with HCMV AD169, or pUL97 kinase-deficient mutant ORF-UL97 K355Δ at MOI of 0.5, or remained-mock infected. 24 h after infection HCMV AD169 infected cells were treated with 0.35 µM (i.e. EC50) pUL97-specific inhibitor maribavir (MBV), or with equal amounts of DMSO. Cells were harvested 3 d p.i. and samples were subjected to standard SDS-PAGE and Wb analysis. Proteins of interest and site-specific phosphorylation were stained using the indicated antibodies. (B) Mean values ± SD of phosphorylation intensity of CDK7 at sites Ser164 and Thr170, as well as CDK9 Thr186, was determined by quadruplicate densitometric quantitation of those Wbs depicted in (A) together with a second biological replicate (shown in Fig. S5). Statistical analysis was performed using an ordinary one-way ANOVA and post-hoc Tukey correction: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., not significant.

Article Snippet: The following antibodies were used in this study: mAb-UL97.01, mAb-UL53, mAb-UL50, and mAb-UL56 (kindly provided by T. Lenac and S. Jonick, University of Rijeka, Croatia), mAb-IE1 (kindly provided by William Britt, Birmingham, AL, USA), mAb-UL44 and mAb-MCP (kindly provided by Bodo Plachter, University of Mainz, Mainz, Germany), mAb-pp65 and mAb-UL69 (kindly provided by T. Stamminger, Ulm, Germany), mAb-β-actin (A5441, Sigma Aldrich), pAb-cyclin H (LSC331195, LS Bio), pAb-CDK7 pSer164 (PA5-105583, Sigma-Aldrich), pAb-CDK7 pThr170 (ab155976, Abcam), pAb-CDK9 pThr186 (2549, Cell signaling), mAb-CDK7 (sc-7344, Santa Cruz Biotechnology), pAbRNAPII CTD pSer2 (NB100-1805, Novus Biologicals), mAb-Flag (F1804, Sigma-Aldrich).

Techniques: Phospho-proteomics, Infection, Activity Assay, Cell Culture, Mutagenesis, SDS Page, Staining, Quantitation Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Novel covalent CDK7 inhibitor potently induces apoptosis in acute myeloid leukemia and synergizes with Venetoclax.

doi: 10.1186/s13046-023-02750-w

Figure Lengend Snippet: Fig. 1 Efficacy of XL102 and its effects on downstream targets of CDK7. A Kinase activity of CDK7 in the presence of XL102 was measured using the LANCE TR-FRET in vitro kinase assay by incubating both CDK7 and XL102 followed by addition of ATP and U-light-MBP peptide substrate. Time-resolved fluorescence (excitation, 320 nm; emission donor, 615 nm; emission acceptor, 665 nm) was monitored by using 2030 multilabel reader Victor5 (PerkinElmer). The IC50 values were derived by fitting a sigmoidal dose–response curve to a plot of assay readout over inhibitor concentration, computed with the Graph Pad Prism. B The pull-down assay using bio-THZ1 show a dose dependent target engagement in AML cells harvested after 3 h of XL102 treatment followed by washing to remove any unbound XL102 (time point-0 h). C XL102 inhibited CTD phosphorylation of conserved residues of RPII in AML cells in dose dependent manner at 6 h and 24 h along with quantification of Western blot data. Results are the mean ± SD of three independent experiments

Article Snippet: Antibodies used against various proteins were CDK7 (Bethyl Laboratories, Massachusetts, US) and Beta actin (Santa Cruz Biotechnology, Texas, US).

Techniques: Activity Assay, In Vitro, Kinase Assay, Fluorescence, Derivative Assay, Concentration Assay, Pull Down Assay, Drug discovery, Phospho-proteomics, Western Blot

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. In theory, acute inhibition of transcriptional CDKs, particularly those involved in phosphorylating RPB1 CTD Ser2 phosphorylation, is expected to globally suppress gene expression, predominantly affecting mRNAs with short half-lives. B. Volcano plots of gene expression derived from 4-6 hours treatment of CDK7 inhibitor THZ1 (250 nM) in ovarian cancer cell line Kuramochi , CDK9 inhibitor HH1 (10 µM) in a cell line (YB5) derived from the SW48 colon cancer cell line , or CDK12 inhibitor SR-4835 (90 nM) in triple-negative breast cancer line MDA-MB-231 . RNA-seq data were downloaded from the Gene Expression Omnibus (GEO) and analyzed. C. A volcano plot of nascent RNA expression from neuroblastoma cells (IMR32) treated with 400 nM THZ531 for 2 hours . Note that the sequencing involved 4-thiouridine-pulse labeling and included RNA spike-in control. D. (Top) selection of TCGA ovarian serous adenocarcinoma samples with low or high expression of the indicated CDK genes (the top and bottom 5% in terms of CDK7, CDK9 or CDK12 mRNA expression in ovarian cancer samples with expression data; n = 17 each group). (Bottom) volcano plots of differential gene expression in tumors with low expressing CDKs compared to those with high expressing CDKs. For all volcano plots, genes significantly upregulated or downregulated (absolute log 2 fold change (FC) ≥ 1, p < 0.1) are colored in red and blue, respectively.

Article Snippet: The following primary antibodies were purchased and used for fluorescence immunoblotting: RNA polymerase II subunit B1 (phospho CTD Ser-2) Antibody, clone 3E10 (EMD Millipore, #04-1571); Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho RNA Polymerase II (S2) Antibody, (Bethyl Laboratories, A300-654A); RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 (EMD Millipore, #04-1572); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523) RNA polymerase II subunit B1 (phospho-CTD Ser-7) Antibody, clone 4E12 (EMD Millipore, #04-1570); RNA Polymerase II Antibody (Bethyl Laboratories, A300-653A); RNA Polymerase II RPB1, clone 8WG16 (BioLegend, #664906); Rpb1 NTD (D8L4Y) Rabbit mAb (Cell Signaling Technology, #14958); c-Myc (clone Y69) Rabbit mAb (Abcam, #ab32072); c-Myc (D84C12) Rabbit mAb (Cell Signaling Technology, #5605); anti-CDK12 (Cell Signaling Technology, #11973); anti-CDK12 (proteintech, #26816-1-AP); anti-CDK12, clone 45F7-H2 (BIO-RAD, #VMA00874); anti-CDK13, clone 46B7-G7 (BIO-RAD, #VMA00875); CDK7 Recombinant Monoclonal Antibody (BL-80-5D4) (Bethyl, #A700-006); CDK7 (MO1) Mouse mAb (Cell Signaling Technology, #2916); CDK8 (D6M3J) Rabbit mAb (Cell Signaling Technology, #17395); CDK9 (C12F7) Rabbit mAb (Cell Signaling Technology, #2316); Anti-CDK19 (Sigma-Aldrich, #HPA007053); JunB Rabbit mAb, clone ARC0268 (ABclonal, #A4848); c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, #9165); c-Fos (9F6) Rabbit mAb (Cell Signaling Technology, #2250); Anti-c-ErbB2/c-Neu (Ab-3) Mouse mAb (3B5) (EMD Millipore, #OP15); anti-PARP (Cell Signaling Technology, #9542); anti-β-Actin, clone AC-15 (Sigma, A5441); anti-Vinculin (Sigma, V9131); anti-β-Tubulin (BioLegend, # 903401).

Techniques: Inhibition, Phospho-proteomics, Gene Expression, Derivative Assay, RNA Sequencing, RNA Expression, Sequencing, Labeling, Control, Selection, Expressing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. HCC1954 cells were treated with THZ531 or NVP-2 for 4 hours, after which whole cell lysates were harvested for fluorescent immunoblotting. The molecular weights of the fluorescent protein markers and clone identities for monoclonal antibodies are indicated. Merged images show signals from two primary antibodies raised in different species. B. Volcano plots of gene expression. HCC1954 cells were treated for 4 h with THZ531 (200 nM), or NVP-2 (200 nM), or vehicle control. Total RNA was extracted and subjected to library construction and deep sequencing. Dotted lines in the volcano plots (left) indicate the thresholds used for log-transformed fold change (1, -1). The Tukey box plots (right) illustrate the comparison between the magnitudes of change in significantly up- or down-regulated genes. **** p < 0.0001; ns, not significant (Mann-Whitney test). C. Analysis of gene size among groups of genes impacted by CDK12 or 9 inhibition. **** p < 0.0001; ns, not significant (Mann-Whitney test). D. Traces of RNA-seq coverage over the gene BLM. Note that CDK12 inhibition decreases coverage over exons, but concomitantly produces reads at positions known to harbor intronic polyadenylation. The boxed regions are also displayed with re-scaled y-axis (bottom). E. Metagene profile plots of genes that are commonly downregulated in HCC1954 cells treated with THZ531, NVP-2, or THZ1 and that have been segmented by gene size. Note that CDK12 inhibition is uniquely associated with a size-dependent elongation defect. F. Overrepresentation analysis of significantly downregulated genes in HCC1954 cells that were treated as indicated in the x-axis. Gene sets in the KEGG and Reactome pathway databases were evaluated. Dot color denotes the statistical significance of gene set enrichment within the group of downregulated genes (-log q) and dot size denotes the fraction of downregulated genes corresponding to that gene set. G. Summary plot of normalized enrichment scores (NES) for gene sets that are significantly downregulated in HCC1954 cells treated with CDK12 inhibitor (nominal p-value < 0.05). Blue dots denote gene sets implicated in the DNA damage response. H. Venn diagram for gene sets that are downregulated by CDK12, CDK9, and CDK7 inhibition. Note that gene sets involved in the DNA damage response are specific to CDK12 inhibition. The right bottom illustrates metagene profile plots of genes in the gene set Reactome_ Homologous DNA Pairing and Strand Exchange in HCC1954 cells treated with THZ531, NVP-2, or THZ1 and that have been segmented by gene size.

Article Snippet: The following primary antibodies were purchased and used for fluorescence immunoblotting: RNA polymerase II subunit B1 (phospho CTD Ser-2) Antibody, clone 3E10 (EMD Millipore, #04-1571); Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho RNA Polymerase II (S2) Antibody, (Bethyl Laboratories, A300-654A); RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 (EMD Millipore, #04-1572); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523) RNA polymerase II subunit B1 (phospho-CTD Ser-7) Antibody, clone 4E12 (EMD Millipore, #04-1570); RNA Polymerase II Antibody (Bethyl Laboratories, A300-653A); RNA Polymerase II RPB1, clone 8WG16 (BioLegend, #664906); Rpb1 NTD (D8L4Y) Rabbit mAb (Cell Signaling Technology, #14958); c-Myc (clone Y69) Rabbit mAb (Abcam, #ab32072); c-Myc (D84C12) Rabbit mAb (Cell Signaling Technology, #5605); anti-CDK12 (Cell Signaling Technology, #11973); anti-CDK12 (proteintech, #26816-1-AP); anti-CDK12, clone 45F7-H2 (BIO-RAD, #VMA00874); anti-CDK13, clone 46B7-G7 (BIO-RAD, #VMA00875); CDK7 Recombinant Monoclonal Antibody (BL-80-5D4) (Bethyl, #A700-006); CDK7 (MO1) Mouse mAb (Cell Signaling Technology, #2916); CDK8 (D6M3J) Rabbit mAb (Cell Signaling Technology, #17395); CDK9 (C12F7) Rabbit mAb (Cell Signaling Technology, #2316); Anti-CDK19 (Sigma-Aldrich, #HPA007053); JunB Rabbit mAb, clone ARC0268 (ABclonal, #A4848); c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, #9165); c-Fos (9F6) Rabbit mAb (Cell Signaling Technology, #2250); Anti-c-ErbB2/c-Neu (Ab-3) Mouse mAb (3B5) (EMD Millipore, #OP15); anti-PARP (Cell Signaling Technology, #9542); anti-β-Actin, clone AC-15 (Sigma, A5441); anti-Vinculin (Sigma, V9131); anti-β-Tubulin (BioLegend, # 903401).

Techniques: Western Blot, Bioprocessing, Gene Expression, Control, Sequencing, Transformation Assay, Comparison, MANN-WHITNEY, Inhibition, RNA Sequencing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. A volcano plot of gene expression in SKBR3 cells treated with THZ531 (200 nM, 4 h) compared with vehicle control (0.04% DMSO, v/v). Blue and red dots denote individual genes with significant change in expression (fold of change > 1, q < 0.1). B. (Left) A Tukey box plot indicates that, for genes significantly altered in expression by THZ531 treatment in SKBR3 cells, the magnitude of upregulation is even greater than gene downregulation (**p < 0.01, Mann-Whitney test). (Right) Comparison of gene size among groups of genes that differentially respond to CDK12 inhibition (****p < 0.0001, Mann-Whitney test). C. HCC1954 cells were treated with vehicle control, 200 nM THZ531 or THZ1 for 4 h, followed by lysis with 1x SDS sample buffer and fluorescent immunoblotting. Note that THZ1 treatment caused a complete electrophoretic mobility shift of total RBP1, indicating a substantial loss of CTD phosphorylation. Note that THZ1 also targets CDK12 and CDK13, and thus lacks a desired selectivity. THZ1 was chosen for the current study, instead of a more selective version of CDK7 inhibitor YKL-5-124, primarily because YKL-5-125 does not have any effect on CTD phosphorylation (Olson et al., 2019). D. A volcano plot of gene expression in HCC1954 cells treated with THZ1 (200 nM, 4 h) compared with vehicle control. Blue and red dots are genes with significant change in expression (fold of change > 1, q < 0.1). E. (Left) A Tukey box plot indicating that, for genes with expression significantly altered by THZ1 treatment in HCC1954 cells, the magnitude of downregulation is greater than gene upregulation (****p < 0.01). (Right) A comparison of gene size among groups of genes that differentially respond to CDK12 inhibition (n.s., not significant; Mann-Whitney test). F. Fluorescent immunoblotting of total cell lysates from HCC1954 cells treated with indicated doses of THZ531 for 4 or 24 hours. Note that cells treated with THZ531 for 24 hours demonstrate a reduced protein abundance of ATM, BRCA1, and EGFR, all of which are encoded by large genes (>100 kb). G. (Top) A schematic depicting mRNA with proximal or distal polyA. (Bottom) The percentage of mRNA in each treatment group (HCC1954) showing positive (proximal polyA) or negative (distal polyA) change of proximal polyadenylation site usage (△PPAU). H. Traces of RNA Seq coverage over the MAP3K9 gene. Note that CDK12 inhibition in HCC1954 cells abolishes reads corresponding to a distal polyA site (boxed) while exerting no effect on coverage over the exons.

Article Snippet: The following primary antibodies were purchased and used for fluorescence immunoblotting: RNA polymerase II subunit B1 (phospho CTD Ser-2) Antibody, clone 3E10 (EMD Millipore, #04-1571); Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho RNA Polymerase II (S2) Antibody, (Bethyl Laboratories, A300-654A); RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 (EMD Millipore, #04-1572); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523) RNA polymerase II subunit B1 (phospho-CTD Ser-7) Antibody, clone 4E12 (EMD Millipore, #04-1570); RNA Polymerase II Antibody (Bethyl Laboratories, A300-653A); RNA Polymerase II RPB1, clone 8WG16 (BioLegend, #664906); Rpb1 NTD (D8L4Y) Rabbit mAb (Cell Signaling Technology, #14958); c-Myc (clone Y69) Rabbit mAb (Abcam, #ab32072); c-Myc (D84C12) Rabbit mAb (Cell Signaling Technology, #5605); anti-CDK12 (Cell Signaling Technology, #11973); anti-CDK12 (proteintech, #26816-1-AP); anti-CDK12, clone 45F7-H2 (BIO-RAD, #VMA00874); anti-CDK13, clone 46B7-G7 (BIO-RAD, #VMA00875); CDK7 Recombinant Monoclonal Antibody (BL-80-5D4) (Bethyl, #A700-006); CDK7 (MO1) Mouse mAb (Cell Signaling Technology, #2916); CDK8 (D6M3J) Rabbit mAb (Cell Signaling Technology, #17395); CDK9 (C12F7) Rabbit mAb (Cell Signaling Technology, #2316); Anti-CDK19 (Sigma-Aldrich, #HPA007053); JunB Rabbit mAb, clone ARC0268 (ABclonal, #A4848); c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, #9165); c-Fos (9F6) Rabbit mAb (Cell Signaling Technology, #2250); Anti-c-ErbB2/c-Neu (Ab-3) Mouse mAb (3B5) (EMD Millipore, #OP15); anti-PARP (Cell Signaling Technology, #9542); anti-β-Actin, clone AC-15 (Sigma, A5441); anti-Vinculin (Sigma, V9131); anti-β-Tubulin (BioLegend, # 903401).

Techniques: Gene Expression, Control, Expressing, MANN-WHITNEY, Comparison, Inhibition, Lysis, Western Blot, Electrophoretic Mobility Shift Assay, Phospho-proteomics, Quantitative Proteomics, RNA Sequencing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. (Left) Summary plot of normalized enrichment scores (NES) for gene sets that are significantly upregulated in HCC1954 cells treated with CDK12 inhibitor (nominal p-value < 0.05). Blue dots denote MYC signature. (Right) GSEA plot of Hallmark_MYC Targets V1 for genes altered by THZ531 (200 nM, 4h) in HCC1954 (top) and SKBR3 (bottom) cells. Normalized enrichment score (NES) and p values are indicated. B. Traces of RNA-seq coverage over MYC in cells that were treated as indicated. Enlarged views for traces of CDK9 and CDK7 inhibition are shown as inserts with the y-axis scaled down. C. HCC1954 cells were treated as indicated for 4 h followed by total RNA extraction and reverse transcription. Quantitative PCR was then performed for the indicated genes. Note that MYC expression is increased, while other select DDR genes demonstrated significant downregulation. *p<0.05, **p<0.01, ***p<0.001 (Student’s t tests). D. HER2+ breast cancer cells were treated with THZ531 (200 nM) for the indicated time points. Cell lysates were prepared in SDS sample buffer and subjected to fluorescent immunoblotting. E. HCC1954 cells were treated with increasing concentrations of THZ531 (0, 12.5, 25, 50,100, 200, 400, and 800 nM) for 4 hours. Cell lysates were prepared and analyzed as in (G). F. HCC1954 cells were treated with Actinomycin D at the indicated doses in the presence of vehicle control or THZ531 (400 nM). Four hours post-treatment, cells were lysed with 1x SDS sample buffer, and lysates were prepared for fluorescent immunoblotting. Merged images are shown for blots using antibodies raised in different species. The very left lane was loaded with protein markers that emit near-infrared fluorescence. G. THZ531-induced MYC expression relies on de novo protein synthesis. HCC1954 cells were treated with cycloheximide (6.25 μg/ml) and THZ531 (200 nM), either individually or in combination. Cells were harvested in 3 hours for immunoblotting. H. Tumors with low expression of CDK12 exhibit activation of a MYC signature. (Right) The consolidated MYC signature was developed by combining all 12 gene sets from MSigDB ( http://www.gsea-msigdb.org/ ) and identifying genes whose expression positively correlates with MYC across the 1305 cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE, Pearson correlation r > 0.2). (Left) Expression of the 235 genes was then evaluated in CDK12, CDK7, CDK9-low ovarian tumors in comparison to their high expression counterparts. The top dot plot displays the differential expression of all 235 genes, while the bottom plot shows genes with significant differential expression (q < 0.1; n = 106, 60, 6 for the CDK12, CDK7, CDK9 groups, respectively). *p<0.05, ****p<0.0001 (Mann-Whitney test).

Article Snippet: The following primary antibodies were purchased and used for fluorescence immunoblotting: RNA polymerase II subunit B1 (phospho CTD Ser-2) Antibody, clone 3E10 (EMD Millipore, #04-1571); Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho RNA Polymerase II (S2) Antibody, (Bethyl Laboratories, A300-654A); RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 (EMD Millipore, #04-1572); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523) RNA polymerase II subunit B1 (phospho-CTD Ser-7) Antibody, clone 4E12 (EMD Millipore, #04-1570); RNA Polymerase II Antibody (Bethyl Laboratories, A300-653A); RNA Polymerase II RPB1, clone 8WG16 (BioLegend, #664906); Rpb1 NTD (D8L4Y) Rabbit mAb (Cell Signaling Technology, #14958); c-Myc (clone Y69) Rabbit mAb (Abcam, #ab32072); c-Myc (D84C12) Rabbit mAb (Cell Signaling Technology, #5605); anti-CDK12 (Cell Signaling Technology, #11973); anti-CDK12 (proteintech, #26816-1-AP); anti-CDK12, clone 45F7-H2 (BIO-RAD, #VMA00874); anti-CDK13, clone 46B7-G7 (BIO-RAD, #VMA00875); CDK7 Recombinant Monoclonal Antibody (BL-80-5D4) (Bethyl, #A700-006); CDK7 (MO1) Mouse mAb (Cell Signaling Technology, #2916); CDK8 (D6M3J) Rabbit mAb (Cell Signaling Technology, #17395); CDK9 (C12F7) Rabbit mAb (Cell Signaling Technology, #2316); Anti-CDK19 (Sigma-Aldrich, #HPA007053); JunB Rabbit mAb, clone ARC0268 (ABclonal, #A4848); c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, #9165); c-Fos (9F6) Rabbit mAb (Cell Signaling Technology, #2250); Anti-c-ErbB2/c-Neu (Ab-3) Mouse mAb (3B5) (EMD Millipore, #OP15); anti-PARP (Cell Signaling Technology, #9542); anti-β-Actin, clone AC-15 (Sigma, A5441); anti-Vinculin (Sigma, V9131); anti-β-Tubulin (BioLegend, # 903401).

Techniques: RNA Sequencing, Inhibition, RNA Extraction, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control, Fluorescence, Activation Assay, Comparison, Quantitative Proteomics, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cryptic splicing in synaptic and membrane excitability genes links TDP-43 loss to neuronal dysfunction

doi: 10.1101/2025.08.28.672801

Figure Lengend Snippet: TDP-43 loss of function directly leads to cryptic splicing and reduced gene expression A. The levels of transcripts with cryptic exons in AKT3 , CACNA1E , CEP290 , KALRN , KCNQ2 , MNAT1 , RAP1GAP , SETD5 , STXBP5L , and SYT7 were increased upon TDP-43 knockdown. qPCR experiments were performed in iNeurons treated with TDP-43 shRNAs for seven days. The level of GAPDH was used for normalization. The level in control condition was set to 1. Mean ± s.e.m., n=3, unpaired t-test, ** p<0.01, *** p<0.001, **** p<0.0001. B. The total transcript levels of AKT3 , CACNA1E , KALRN , KCNQ2 , RAP1GAP , SETD5 , and SYT7 were decreased upon TDP-43 knockdown while the total transcript level of STXBP5L was increased. qPCR experiments were performed in iNeurons treated with TDP-43 shRNAs for seven days. The level of GAPDH was used for normalization. The level in control condition was set to 1. Mean ± s.e.m., n=3, unpaired t-test, ** p<0.01, *** p<0.001, **** p<0.0001. C. The total protein levels of UNC13A , KALRN , MNAT1 , RAP1GAP , and SYT7 were decreased upon TDP-43 knockdown. Western blotting experiments were performed in iNeurons treated with TDP-43 shRNAs for twelve days. The level of GAPDH or Tubulin was used for normalization. The level in control condition was set to 1. Mean ± s.e.m., n=3, unpaired t-test, *** p<0.001, **** p<0.0001. D. TDP-43 binding sites or UG-rich motifs were detected in the cryptic splicing region of KALRN , KCNQ2 , MNAT1 , RAP1GAP , SETD5 , and SYT7 . Lane 1, RNA-seq track of TDP-43 positive nuclei from FTD/ALS patient brain tissues; Lane 2, RNA-seq track of TDP-43 negative nuclei from FTD/ALS patient brain tissues; Lane 3, CLIP-seq track of TDP-43 in SH-SY5Y cells; Lane 4, percentage of UG (TG) or GU (GT) dinucleotides in 20bp bins, displayed as IGV tracks with a data range of 0.4–1; red rectangle, cryptic exon.

Article Snippet: Primary antibodies used in this study were: TDP-43 (Proteintech, 10782-2-AP), Tubulin (Cell Signaling Technology, 2144S), GAPDH (Sigma-Aldrich, G8795), UNC13A (Proteintech, 68483-1-Ig), KALRN (Proteintech, 19740-1-AP), RAP1GAP (Abcam, ab32373), SYT7 (Thermo Scientific, PA5-52998), MNAT1 (Proteintech, 11719-1-AP).

Techniques: Gene Expression, Knockdown, Control, Western Blot, Binding Assay, RNA Sequencing

Journal: bioRxiv

Article Title: Cryptic splicing in synaptic and membrane excitability genes links TDP-43 loss to neuronal dysfunction

doi: 10.1101/2025.08.28.672801

Figure Lengend Snippet: Cryptic splicing targets of TDP-43 are critical for maintaining neuronal activity A. Schematic diagrams show the experimental design of multielectrode array (MEA) recordings upon knockdown of TDP-43 or its cryptic splicing targets in iNeurons in two batches. Batch 1 examined the effects of a reduction in TDP-43, KALRN, RAP1GAP, SYT7, and UNC13A, and Batch 2 examined the effects of a reduction in TDP-43, KCNQ2, MNAT1, and STMN2. Knockdown or scramble shRNAs were administered on Day 21 in both batches. A first evaluation was conducted on Day 40 in Batch 1 and on Day 30 in Batch 2 (T1). A second evaluation was conducted on Day 48 in Batch 1 and on Day 35 in Batch 2 (T2). B. MEA analysis shows decreases in the weighted mean firing rate (spontaneous firing) and the number of bursts (excitability) upon reductions in TDP-43, RAP1GAP, SYT7, KCNQ2, and MNAT1 at T1, as well as decreases in the synchrony index (connectivity) upon reductions in TDP-43, KALRN, RAP1GAP, SYT7, UNC13A, KCNQ2, and MNAT1 at T2. Mean ± s.e.m., n=8, one-way ANOVA, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Primary antibodies used in this study were: TDP-43 (Proteintech, 10782-2-AP), Tubulin (Cell Signaling Technology, 2144S), GAPDH (Sigma-Aldrich, G8795), UNC13A (Proteintech, 68483-1-Ig), KALRN (Proteintech, 19740-1-AP), RAP1GAP (Abcam, ab32373), SYT7 (Thermo Scientific, PA5-52998), MNAT1 (Proteintech, 11719-1-AP).

Techniques: Activity Assay, Knockdown

Journal: Frontiers in Oncology

Article Title: PKM2 Interacts With the Cdk1-CyclinB Complex to Facilitate Cell Cycle Progression in Gliomas

doi: 10.3389/fonc.2022.844861

Figure Lengend Snippet: PKM2 physically interacts with the Cdk1-cyclin B complex. (A) Levels (top) and activity (bottom) of Cdk7 in control and PKM2 knock-down U87, T98G and LN319 cells 10 hrs following release from serum deprivation-induced arrest. (B) Levels of cyclin B1, PKM2, and Cdk1 in total cell lysates in control or PKM2 shRNA U87 cells measured at the times indicated following release from serum deprivation-induced arrest. (C) Levels of Cdk1, cyclin B, or PKM2 from Cdk1, cyclin B or PKM2 immunoprecipitates measured at the times indicated following release of control or PKM2 shRNA U87 cells from serum deprivation-induced arrest. Data in M lanes were derived from cells isolated by mitotic shake-off. I:B = Immuno Blot, IP = immunoprecipitate. Bottom panel: Densitometric analysis of Cdk1, cyclin B, or PKM2 levels in U87 cells from Cdk1, cyclin B or PKM2 immunoprecipitates. (D) Western blot analysis of cyclin B1 in control and PKM2 knock-down U87, T98G and LN319 cells. Densitometric analysis (bottom right panel) of western blot demonstrating levels of cyclin B1 in different experimental groups. *p<.05, n=3.

Article Snippet: For Cdk1-cyclin B activity analysis, cell lysates were diluted and pipetted into recombinant Cdc7 pre-coated wells (MBL International Corp), after which Mg2+ and ATP were added, and the amount of phosphorylated substrate was measured by binding to an anti-phospho-Cdk7 (T 376) antibody and a horseradish peroxidase-conjugated anti-mouse IgG.

Techniques: Activity Assay, Control, Knockdown, shRNA, Derivative Assay, Isolation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: CDKN1C Negatively Regulates RNA Polymerase II C-terminal Domain Phosphorylation in an E2F1-dependent Manner *

doi: 10.1074/jbc.M109.091496

Figure Lengend Snippet: CDKN1C interacts with CDK7 and 9 in vivo. A, H1299 cells were co-transfected with the indicated expression vectors or empty vector. IP/Western blots were performed using CDKN1C antibody sc-1040 (Santa Cruz Biotechnology) for both IP and Western blot. The FLAG antibody F-1804 from Sigma was used for detection of tagged CDK7 and CDK9 by Western blot. An anti-FLAG antibody conjugated to agarose (A-2220, Sigma) was used for FLAG-IPs. B–D, HeLa cells extracts (with and without prior dexamethasone (Dex) treatment as indicated) were subjected to IP/Western blots using the indicated combinations of antibodies. Results reveal that endogenous CDK 7associates with both E2F1 and CDKN1C and that CDKN1C interacts with RNA pol II in vivo.

Article Snippet: Cleared supernatants were immunoprecipitated with anti-CDK7 antibody (BD Pharmingen) or anti-CDK9 antibody (Cell Signaling Technology) and protein A/G-agarose, the beads were washed with TGN buffer followed by TGN buffer plus 0.5 m LiCl, and two washes with kinase buffer (20 m m HEPES, pH 7.5, 50 m m NaCl, 10 m m MgCl 2 , and 1 m m dithiothreitol).

Techniques: In Vivo, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: CDKN1C Negatively Regulates RNA Polymerase II C-terminal Domain Phosphorylation in an E2F1-dependent Manner *

doi: 10.1074/jbc.M109.091496

Figure Lengend Snippet: CDKN1C can inhibit CDK7 and CDK9 kinase activity toward the RNA pol II CTD. A, CDK9 was immunopurified from extracts of control H1299 cells or H1299 cells infected with Ad-p57 and subjected to kinase assays using bacterially produced GST-CTD as substrate. A single “+” sign indicates that 250 μg of cell extract was used for IP, whereas “++” indicates 500 μg was used. Following 30-min incubations, reactions were subjected to SDS-PAGE and Western blotting using the indicated antibodies. The Western blot signal using phospho-serine specific antibodies indicated the relative level of kinase activity toward CTD. B, CDK7 was immunoprecipitated from 30 μg of cell extract, and the kinase reaction was carried out for 20 min. C, H1299 cells were infected for 48 h with the indicated combination of viral vectors to express cdkn1c (p57) or CDK7. Overexpression of CDK7 rescues CTD-Ser-2 phosphorylation in the presence of excess cdkn1c. A similar viral vector for CDK9 was not available.

Article Snippet: Cleared supernatants were immunoprecipitated with anti-CDK7 antibody (BD Pharmingen) or anti-CDK9 antibody (Cell Signaling Technology) and protein A/G-agarose, the beads were washed with TGN buffer followed by TGN buffer plus 0.5 m LiCl, and two washes with kinase buffer (20 m m HEPES, pH 7.5, 50 m m NaCl, 10 m m MgCl 2 , and 1 m m dithiothreitol).

Techniques: Activity Assay, Infection, Produced, SDS Page, Western Blot, Immunoprecipitation, Over Expression, Plasmid Preparation

Journal: Cell Proliferation

Article Title: Role of protein kinase C‐iota in transformed non‐malignant RWPE‐1 cells and androgen‐independent prostate carcinoma DU‐145 cells

doi: 10.1111/j.1365-2184.2009.00582.x

Figure Lengend Snippet: Association of PKC‐ι, with Cdk7 in RWPE‐1 cells. Whole cell extracts (1 mg) from each time point were immunoprecipitated with rabbit polyclonal anti‐Cdk7 (5 µg) as described in the Materials and methods section. Column 1 contains both positive (+) and two negative controls (–). Positive control (+) is the whole cell lysates (100 µg); the first negative control (–) contains whole cell lysates (1 mg) plus rabbit IgG whole molecule (50 µl of 1 : 1 v/v) and the second negative control contains whole cell lysate (1 mg) plus rabbit IgG whole molecule (50 µl) and normal rabbit IgG serum (5 µg). Column 2 is cell lysates taken at indicated time points. Immunoprecipitates were separated by SDS‐PAGE and were Western blotted with anti‐PKC‐ι mouse monoclonal antibody. Physical association of PKC‐ι and Cdk7 were observed at 30 h time points (a). Immunoprecipitation (IP) with rabbit polyclonal Cdk7 (b) showed that Cdk2 is also coimmunoprecipitated (c). Western blotting of phospho‐Cdk7 (p‐Cdk7; T170) (d) and phospho‐Cdk2 (p‐Cdk2; T160) were also observed (e). Presence of Cdk2 was observed throughout the cell cycle (f) and β‐actin (g) shows equal loading of samples in each lane.

Article Snippet: Reagents and antibodies Primary antibodies were purchased from the following companies: PKC‐α, βΙ, δ, ɛ, γ, θ (Santa Cruz Biotechnology, Santa Cruz, CA, USA); PKC‐ι (Transduction Laboratory, Lexington, KY, USA); PKC‐ζ (Upstate Biotechnology, Lake Placid, NY, USA); p‐Bad (ser‐112), p‐Bad (ser‐155), p‐Bad (ser‐136), Bad, PARP, cytochrome c, caspase‐7, survivin, Cdk2, Cdk7, β‐actin (Santa Cruz Biotechnology); cleaved PARP (Asp 214), Bcl‐x L , p‐Cdk2 (Ther160) (Cell Signalling Technology, Danvers, MA, USA); and phospho‐Cdk7‐T170 (Abgent, San Diego, CA, USA).

Techniques: Immunoprecipitation, Positive Control, Negative Control, SDS Page, Western Blot

Journal: Cell Proliferation

Article Title: Role of protein kinase C‐iota in transformed non‐malignant RWPE‐1 cells and androgen‐independent prostate carcinoma DU‐145 cells

doi: 10.1111/j.1365-2184.2009.00582.x

Figure Lengend Snippet: PKC‐ι siRNA leads to apoptosis. Whole cell extracts of RWPE‐1 treated with control siRNA and PKC‐ι siRNA were prepared as described in the Materials and methods section and immunoblot analysis of PARP and cleaved PARP (Asp214), indicated cells are undergoing apoptosis via activation of caspase‐7. Immunoblotting for survivin and cytochrome c further demonstrated apoptosis in PKC‐ι siRNA treated cells. Western blot analysis of β‐actin shows that an equal amount of protein was loaded in each lane. Similar immunoblots were performed for DU‐145 cells. Activation of PARP and caspase‐7, combined with an increase in cytochrome c and decrease in survivin indicated apoptosis in DU‐145 cells. (b) A separate experiment was repeated for RWPE‐1 cells with control siRNA and PKC‐ι siRNA and whole cells extracts (150 µg) were analysed for Cdk7, p‐Cdk7 (T170), Cdk2, p‐Cdk2 (T160), and β‐actin to verify equal loading of protein. (c) Densitometry for p‐Cdk7 and p‐Cdk2 showed a significant decrease in PKC‐ι siRNA‐treated cells compared to control siRNA (P = 0.001 and 0.035, respectively).

Article Snippet: Reagents and antibodies Primary antibodies were purchased from the following companies: PKC‐α, βΙ, δ, ɛ, γ, θ (Santa Cruz Biotechnology, Santa Cruz, CA, USA); PKC‐ι (Transduction Laboratory, Lexington, KY, USA); PKC‐ζ (Upstate Biotechnology, Lake Placid, NY, USA); p‐Bad (ser‐112), p‐Bad (ser‐155), p‐Bad (ser‐136), Bad, PARP, cytochrome c, caspase‐7, survivin, Cdk2, Cdk7, β‐actin (Santa Cruz Biotechnology); cleaved PARP (Asp 214), Bcl‐x L , p‐Cdk2 (Ther160) (Cell Signalling Technology, Danvers, MA, USA); and phospho‐Cdk7‐T170 (Abgent, San Diego, CA, USA).

Techniques: Western Blot, Activation Assay