recombinant proteins sr 4835 medchemexpress  (MedChemExpress)

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 CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

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. Perturbing CDK12 in breast cancer cells by CRISPR-mediated gene editing results in gene upregulation as well as downregulation. In contrast, knocking down CDK13 primarily leads to gene downregulation B. Inducible knockdown of Ccnk in mouse leukemia cells , the cyclin for CDK12 and CDK13, results in profound upregulation of gene expression. C. In mouse embryonic stem cells with ablated endogenous Cdk12 alleles, repressing the expression of transgenic Cdk12 induces both downregulation and upregulation of gene expression. D. Silencing CDK12 expression with siRNA in HER2+ (SKBR3) or triple negative breast cancer cell line (MDA-MB-231), as well as a non-transformed breast cell line (184-hTERT) , results in dual-modal alteration of global gene expression. All RNA-seq data were obtained from the NCBI Gene Expression Omnibus (GEO) and analyzed as described in the Methods section. Information regarding experimental procedures is from the original publications , , . In the volcano plots, genes significantly upregulated or downregulated (absolute log 2 fold change ≥ 1, p < 0.1) are colored in red and blue, respectively.

Article Snippet: The CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

Techniques: CRISPR, Knockdown, Gene Expression, Expressing, Transgenic Assay, Transformation Assay, RNA Sequencing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. T-SNE plots for TCGA breast cancer (top), prostate caner (middle), and ovarian cancer (bottom) samples, each clustered according to PAM50 gene signature. All three plots are on the same x- and y-axis scales. The insert in the ovarian plot provides a zoomed-in view. Note that compared with breast cancer, ovarian cancer has a more homogenous expression of PAM50 genes, and that unlike prostate cancer, the CDK12 low or high ovarian samples are more dispersed out rather than clustered together. B. (top) A volcano plot comparing gene expression in ovarian tumors with CDK12 deletion or loss-of-function mutations to tumors with high expression of CDK12. (below) The table lists all TCGA ovarian tumors samples with CDK12 gene alterations.

Article Snippet: The CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

Techniques: Expressing, Gene Expression

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. Gene copy number status of human chromosome 17 (GDC TCGA breast cancer dataset). The heatmap was generated using the UCSC Xena Functional Genomics Browser (xena.ucsc.edu). Note that the HER2/ERBB2 amplicon occurs in approximately 15% breast cancer patients. B. A diagram depicting the structure of the HER2/ERBB2 amplicon in human chromosome 17. C. Association between the expression of HER2/ERBB2 amplicon genes and tumor grade. Correlation analysis was performed on gene expression in relation to the histologic grade of HER2+ breast cancer of the METABRIC cohort , with p values generated from One-way ANOVA analysis. (Left) Plot for CDK12 gene expression in HER2+ tumors of different histological grades. (Right) A summary of the statistical significance for the association. D. The indicated cells or primary culture were treated with vehicle control or THZ531 (100 nM) for seven days, followed by fixation and crystal violet staining (left), and cell growth quantification (right). Note that DFBM-NI17 cells, which grow in suspension, had their cell growth measured in 96-well plates using CellTiter-Glo assays. E. HER2+ breast cancer cells (SKBR3) were transduced with lentiCRISPR virus targeting the indicated genes. After puromycin selection, cells 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 antibodies raised in different species. F. Cells as in (D) were seeded in 12-well plates at a density of 5,000 cells per well. After one week of incubation, cells were fixed and stained with crystal violet. The staining was subsequently solubilized for quantification of cell growth. ***p < 0.001. G. Mice with orthotopic xenografts of HER2+ breast cancer cells (HCC1954) were treated with SR-4835 (20 mg/kg by gavage) or vehicle (30% hp-BCD). Tumor size is shown as mean ± SE. H. Tumor size measurement of individual HCC1954 tumors over time (fine lines) and fitted tumor volume estimates (bold lines). Dashed red line in the right plot represents the fitted tumor growth curve from mice treated with vehicle. I. Kaplan-Meier curve demonstrating the percentage of animals with xenograft HER2+ tumors (HCC1954) smaller than 500 mm at the indicated time (p < 0.0001, log-rank [Mantel-Cox] test).

Article Snippet: The CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

Techniques: Generated, Functional Assay, Amplification, Expressing, Gene Expression, Control, Staining, Suspension, Transduction, Virus, Selection, Western Blot, Bioprocessing, Incubation

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. An example showing that genes within the HER2/ERBB2 amplicon could have vastly different patterns of expression. The left dot plot indicates a nearly perfect correlation between the copy number of ERBB2 and that of IKZF3, the middle dot plot depicts a statistically weak correlation between the gene expression of ERBB2 and IKZF3, and the right dot pot shows a lack of correlation between the protein abundance of ERBB2 and IKZF3. Gene copy number and expression (RNA-seq) data in the GDC TCGA breast cancer cohort and proteomic data were analyzed in GraphPad Prism. The Pearson correlation coefficient ( r ) is indicated. B. Expression of indicated genes in HER2 negative (HER2-) or HER2 positive (HER2+) breast tumors. The analysis was performed via Oncomine ( www.oncomine.org ; ) for the cohorts of Kao_breast and TCGA_breast (Cancer Genome Atlas Network et al., 2012). The statistical comparison between HER2+ and HER2-tumors include FC (fold change of gene expression), p value, and genome-wide ranking that is based on p values. C. Analysis of CDK12 gene expression in relation to the histologic grade of breast cancer in total cohort of breast cancer. CDK12 gene expression and the annotation of histologic grade of tumors were generated in the Curtis cohort . The indicated p values were generated from One-way ANOVA analysis. D. Expression of CDK12 in normal breast tissues, ER/PR+, triple-negative, or HER2+ breast cancers. The expression data were generated in the Curtis (METABRIC) or TCGA cohort . E. (Left) Abundance of indicated proteins in human breast cancer samples. The oncoprint visualization was derived from a query into the dataset of TCGA Breast Invasive Carcinoma at cBioPortal for Cancer Genomics ( www.cbioportal.org ; ). A total of 74 samples were subjected to mass-spectrometry-based proteomic analysis . (Right) CDK12 protein abundance in breast cancer is significantly correlated with that of ERBB2. Quantification data were downloaded from cBioPortal and analyzed in GraphPad Prism 7. The Pearson correlation coefficient ( r ) is indicated.

Article Snippet: The CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

Techniques: Amplification, Expressing, Gene Expression, Quantitative Proteomics, RNA Sequencing, Comparison, Genome Wide, Generated, Derivative Assay, Mass Spectrometry

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. The anti-proliferative effects of the HER2 kinase inhibitor Lapatinib (top) and the CDK12 inhibitor THZ531 (bottom) in HER2+ breast cancer cell lines (black curves). Note that the ER+ breast cancer cell line ZR-75-1 (blue curve) showed resistance to Lapatinib while exhibiting modest sensitivity to THZ531. B. HER2+ breast cancer cells were seeded in 12-well plates and treated with vehicle control (DMSO) or THZ531 (50 nM for BT474 and ZR-75-30; 200 nM for HCC1954). Upon reaching visual confluency in the group of vehicle treatment, cells were imaged using a 10x objective lens (left). Cells were then fixed and stained with crystal violet. C. Induction of apoptotic cell death by THZ531. HER2+ breast cancer cells were treated with increasing concentrations of THZ531 for 24 h, unless specified otherwise. Cell lysates were prepared for fluorescent immunoblotting. The molecular weights of the fluorescent protein markers are indicated. D. Response of HER2+ breast cancer cells to T-DM1. The indicated HER2+ cancer cell lines and primary cultures of HER2+ metastatic breast cancer were exposed to T-DM1 (100 ng/ml for HCC1954, SKBR3, and DFBC-1407; 20 ng/ml for DFBM-NI17). Images were acquired after seven days of incubation using a 10x objective lens. E. Cells were treated as indicated, fixed, and stained with crystal violet (top). The staining was subsequently extracted for quantification of cell growth (bottom). Note that the triple-negative breast cancer cells (MDA-MB-468) were used as a negative control and demonstrated complete resistance to T-DM1. F. HER2+ breast cancer cell lines and primary cultures of HER2+ metastatic breast cancer were treated with vehicle control (0.02% DMSO, v/v) or THZ531 (100 nM). After seven days, bright-field images were captured using a 10x objective lens. G. HER2+ breast cancer cells (SKBR3) were transduced with lentiCRISPR virus targeting the indicated genes. After puromycin selection, cells were subjected to seven-day cell proliferation assays measured by CellTiter-Glo. Raw luminescence values of each group were plotted (mean ± SD). Unpaired Student’s t tests revealed significant differences between sgGFP and all sgCDK groups (p < 0.0001), except sgCDK19. Note that sgCDK12 cells demonstrated the most impaired cell proliferation. H. Bright-field images of SKBR3 cells transduced with indicated guides as in (G). Cells were seeded in 12-well plates at a density of 5,000 cells per well. After one week of incubation, images were captured using a 10x objective lens.

Article Snippet: The CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

Techniques: Control, Staining, Western Blot, Incubation, Negative Control, Transduction, Virus, Selection

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 CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

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 CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

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. Overrepresentation analysis of significantly downregulated (left) or upregulated (right) genes in each of the treatments. Gene sets from the GO term database (> 10,000 gene sets) were evaluated. Dot color indicates the statistical significance (-log q) of the gene set representation within the group of down- or upregulated genes, and dot size represents the fraction of down- or upregulated genes corresponding to each gene set represented. B. Normalized enrichment score (NES) plots for gene sets that are significantly upregulated (left) or downregulated (right) in SKBR3 cells following CDK12 inhibition (nominal p-value< 0.05). Two MYC gene sets are highlighted and indicated (left), and 4 gene sets implicated in DNA damage response are highlighted in blue (right). C. GSEA plots for indicated gene sets in HCC1954 (top) and SKBR3 (bottom) cells treated with THZ531. NES and p values are indicated. D. NES plot for gene sets that are significantly downregulated in HCC1954 cells treated with the CDK9 inhibitor NVP-2 (nominal p-value < 0.05). The blue dots denotes MYC signature. The right plots depicts GSEA analysis of Hallmark_MYC Targets V2 gene set for genes downregulated by NVP-2 (200 nM, 4 h; top) and THZ1 (200 nM, 4 h; bottom). NES and p values are indicated.

Article Snippet: The CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

Techniques: Inhibition

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 CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

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: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. The indicated breast cancer cell lines and non-transformed human cells were treated with either DMSO vehicle control or THZ531 (200, 400 nM) for 4 h. Cells were then lysed using 1x SDS sample buffer and subjected to fluorescent immunoblotting. B. HER2+ breast cancer cells (HCC1954) and triple-negative breast cancer cells (MDA-MB-468) were treated with increasing doses of THZ531 for 4 h, followed by fluorescent immunoblotting. Note that FOS expression is induced in both cell lines, while MYC is only induced in HER2+ breast cancer cells. C. A volcano plot illustrates gene expression changes in triple-negative breast cancer cells (MDA-MB-468) treated with 200 nM THZ531 (4 h), compared with cells treated with vehicle control. Note that, despite the induction of a large number of genes, MYC expression is not altered by THZ531 treatment. D. A summary of MYC gene expression alteration following short-term exposure to CDK12 inhibitors. Note that robust MYC induction by CDK12 inhibition was also observed in the primary cultures of HER2+ metastatic breast tumor DFBC-1407

Article Snippet: The CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

Techniques: Transformation Assay, Control, Western Blot, Expressing, Gene Expression, Inhibition

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. MYC expression in parental HCC1954 cells and in HCC1954 cells following the infection of MYC-targeting lentiCRISPR vector (sg_MYC). The cells were treated with vehicle or THZ531 (200 nM) for 4 hours, followed by lysate preparation and fluorescent immunoblotting. B. Dot plot showing that treated sgMYC cells have similar global gene expression to treated parental cells. C. MYC signature is the top-ranked gene set among those downregulated in sgMYC cells treated with THZ531 relative to the treated, parental controls. Summary plot of normalized enrichment scores (NES) for gene sets that are significantly downregulated in sgMYC HCC1954 cells treated with CDK12 inhibitor (nominal p-value < 0.05). Blue dots denote MYC signatures. D. Expression of the consolidated 235-gene MYC signature in sgMYC HCC1954 cells treated with THZ531 compared to the treated, parental controls. The first column shows all genes, while the second shows the differentially regulated genes (DEGs, q<0.1). E. Response of control and sg_MYC cells to THZ531. The cells were treated with THZ531 at the indicated concentrations and, in 9 days, fixed for crystal violet staining (top) and subsequent quantification (bottom). F. CellTiter-Glo luminescent cell viability assays of parental and sgMYC cells treated with increasing concentrations of THZ531 (top) or doxorubicin (bottom) for 7 days. G. Heatmaps of ERBB2, CDK12, CDK13 and MYC expression in normal and malignant breast tissues. The data were generated with the use of UCSC Xena Functional Genomics Browser. Note that HER2+ breast tumors (highlighted) exhibit high expression of HER2/ERBB2 and CDK12, but lower expression of MYC compared to normal breast tissues. H. Plot of ERBB2 and MYC expression in breast tumor samples (TCGA). The Pearson correlation coefficient ( r ) and p value were calculated in GraphPad Prism 7. I. Reverse correlation between ERBB2 and MYC expression in lung cancer samples (TCGA). J. Fluorescent immunoblotting for analysis of MYC and cleaved PARP. Cells were infected with lentivirus, and harvested six days after the initial virus infection for the preparation of lysates. Note that apoptotic cell death, indicated by the appearance of cleaved PARP, occurs upon forced MYC overexpression in two HER2+ breast cancer cells, but is negligible in triple-negative MDA-MB-231 cells. K. Effect of MYC overexpression on the growth of cancer cells. The cells were transduced wit freshly packaged virus and, on day 5, seeded in 12-well plates (5,000 MDA-MB-231 cells, 20,000 HCC1954 or SKBR3 cells seeded in each well). Cells were fixed in 6 – 8 days upon reaching visual confluence and stained with crystal violet (left). The staining was then dissolved for the quantification of cell growth (right).

Article Snippet: The CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

Techniques: Expressing, Infection, Plasmid Preparation, Western Blot, Gene Expression, Control, Staining, Generated, Functional Assay, Virus, Over Expression

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: (A) The indicated MYC-mediated repression signature is upregulated in sgMYC cells treated with THZ531 compared to similarly treated parental controls. Summary plot of normalized enrichment scores (NES) for gene sets that are significantly upregulated in sgMYC HCC1954 cells treated with CDK12 inhibitor (nominal p-value < 0.05). (B) GSEA plots of indicated signatures in sgMYC HCC1954 cells treated with THZ531 (4 h, 200 nM) versus parental cells treated with THZ531. Normalized enrichment score (NES) and p values are indicated. (C) GSEA plots as in (B) for sgMYC HCC1954 cells in comparison to the parental controls. MYC signature is negatively enriched in sgMYC cells. (D) Expression analysis of the consolidated 235-gene MYC signature in sgMYC HCC1954 cells compared to parental controls. The first column shows the relative expression of all 235 genes, while the second column displays differentially regulated genes (DEGs, q < 0.1). (E) Overrepresentation analysis of GO_BP terms in the set of genes upregulated upon treatment with THZ531 in sgMYC HCC1954 cells compared to treated parental controls. Note that terms associated with the positive regulation of cell proliferation and the negative regulation of apoptosis are enriched. (F) Workflow outlining the construction of MYC knockout cancer cells. The HER2+ cancer line BT474 was transduced with sgMYC and subsequently seeded with parental cells at a low density. Puromycin was added in two weeks to eliminate parental cells. Outgrown clones were picked for expansion and validated by immunoblotting. Note that #9 clone in the blot was chosen as a knockout clone based on the absence of signals corresponding to the p64 and p67 forms of MYC . (G) MYC knockout clone (#9) and parental BT474 cells were treated with increasing concentrations of THZ531 in 96-well plates for 9 days before CellTiter-Glo assays.

Article Snippet: The CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

Techniques: Comparison, Expressing, Knock-Out, Transduction, Clone Assay, Western Blot

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. MYC and ERBB2 gene expression data from the METABRIC cohort were obtained from Oncomine and analyzed using GraphPad Prism. The Pearson correlation coefficient ( r ) is indicated. B. Heatmap of ERBB2 and MYC expression in TCGA Lung Cancer dataset. The data were generated using the UCSC Xena Functional Genomics Browser ( http://xena.ucsc.edu/ ). (C-G). Heatmaps and dot plots of ERBB2 and MYC expression in TCGA datasets for Bladder Cancer (C), Cervical Cancer (D), Kidney Clear Cell Carcinoma (E), Kidney Papillary Cell Carcinoma (F), and Prostate Cancer (G). RNA-seq data were obtained from the UCSC Xena Functional Genomics Browser and visualized using GraphPad Prism. H. Expression of MYC, CDK12, and ERBB2 in HER2+ breast cancer, compared to those in normal breast tissues. The gene expression data were generated by METABRIC cohort ( 51 ). Black lines in each group indicate median with interquartile range. I. (top) An overview of single-cell RNA-seq data generation and analysis. Single-cell RNA seq data of breast cancer from Wu et al. study were analyzed using the Broad Institute’s Single-Cell Portal ( https://singlecell.broadinstitute.org/single_cell ). (bottom) Heatmaps display the expression of indicated genes across various cell types in breast tumor samples. Note that epithelial cells within HER2+ breast cancer demonstrate the lowest expression of MYC and, as expected, the highest expression of ERBB2 and CDK12.

Article Snippet: The CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

Techniques: Gene Expression, Expressing, Generated, Functional Assay, RNA Sequencing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. The anti-proliferative effects of the HER2 kinase inhibitor Lapatinib (top) and the CDK12 inhibitor THZ531 (bottom) in HER2+ breast cancer cell lines (black curves). Note that the ER+ breast cancer cell line ZR-75-1 (blue curve) showed resistance to Lapatinib while exhibiting modest sensitivity to THZ531. B. HER2+ breast cancer cells were seeded in 12-well plates and treated with vehicle control (DMSO) or THZ531 (50 nM for BT474 and ZR-75-30; 200 nM for HCC1954). Upon reaching visual confluency in the group of vehicle treatment, cells were imaged using a 10x objective lens (left). Cells were then fixed and stained with crystal violet. C. Induction of apoptotic cell death by THZ531. HER2+ breast cancer cells were treated with increasing concentrations of THZ531 for 24 h, unless specified otherwise. Cell lysates were prepared for fluorescent immunoblotting. The molecular weights of the fluorescent protein markers are indicated. D. Response of HER2+ breast cancer cells to T-DM1. The indicated HER2+ cancer cell lines and primary cultures of HER2+ metastatic breast cancer were exposed to T-DM1 (100 ng/ml for HCC1954, SKBR3, and DFBC-1407; 20 ng/ml for DFBM-NI17). Images were acquired after seven days of incubation using a 10x objective lens. E. Cells were treated as indicated, fixed, and stained with crystal violet (top). The staining was subsequently extracted for quantification of cell growth (bottom). Note that the triple-negative breast cancer cells (MDA-MB-468) were used as a negative control and demonstrated complete resistance to T-DM1. F. HER2+ breast cancer cell lines and primary cultures of HER2+ metastatic breast cancer were treated with vehicle control (0.02% DMSO, v/v) or THZ531 (100 nM). After seven days, bright-field images were captured using a 10x objective lens. G. HER2+ breast cancer cells (SKBR3) were transduced with lentiCRISPR virus targeting the indicated genes. After puromycin selection, cells were subjected to seven-day cell proliferation assays measured by CellTiter-Glo. Raw luminescence values of each group were plotted (mean ± SD). Unpaired Student’s t tests revealed significant differences between sgGFP and all sgCDK groups (p < 0.0001), except sgCDK19. Note that sgCDK12 cells demonstrated the most impaired cell proliferation. H. Bright-field images of SKBR3 cells transduced with indicated guides as in (G). Cells were seeded in 12-well plates at a density of 5,000 cells per well. After one week of incubation, images were captured using a 10x objective lens.

Article Snippet: The CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

Techniques: Control, Staining, Western Blot, Incubation, Negative Control, Transduction, Virus, Selection

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 CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

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. THZ531, but not inhibitors targeting CDK8 (CCT251545), or CDK9 (NVP-2), or BET family of bromodomain proteins (JQ1) increases MYC expression. SKBR3 cells were treated with indicated inhibitors (200 nM) for 4 hours, followed by fluorescent immunoblotting. B. HCC1954 cells were treated with indicated inhibitors (all 200 nM, except 500 nM for flavopiridol) for 4 hours, followed by fluorescent immunoblotting. C. HCC1954 cells were treated with NVP-2 for 4 hours at indicated concentrations. Note that NVP-2 does not obviously induce MYC expression at low doses, and totally abolished MYC expression at 40 or 200 nM. D. HCC1954 cells were treated with increasing concentrations of flavopiridol for 4 hours. Lysates were prepared and subjected to fluorescent immunoblotting. E. HCC1954 were treated with vehicle (0.08% DMSO, v/v), CDK8/19 inhibitor CCT251545 at the indicated concentrations, or THZ531 (400 nM). Four hours post treatment, cells were lysed with 1x SDS sample buffer, and cell lysates were subjected to fluorescent immunoblotting using the indicated antibodies. 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. Note that for all immunoblotting based on short duration of treatment, cells were lysed with the same amount of sample buffer for complete lysis, and the same volume of lysates was loaded onto each lane of SDS-PAGE gels. Thus, each lane represents signal from similar number of cells.

Article Snippet: The CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

Techniques: Expressing, Western Blot, Bioprocessing, Lysis, SDS Page

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. The indicated breast cancer cell lines and non-transformed human cells were treated with either DMSO vehicle control or THZ531 (200, 400 nM) for 4 h. Cells were then lysed using 1x SDS sample buffer and subjected to fluorescent immunoblotting. B. HER2+ breast cancer cells (HCC1954) and triple-negative breast cancer cells (MDA-MB-468) were treated with increasing doses of THZ531 for 4 h, followed by fluorescent immunoblotting. Note that FOS expression is induced in both cell lines, while MYC is only induced in HER2+ breast cancer cells. C. A volcano plot illustrates gene expression changes in triple-negative breast cancer cells (MDA-MB-468) treated with 200 nM THZ531 (4 h), compared with cells treated with vehicle control. Note that, despite the induction of a large number of genes, MYC expression is not altered by THZ531 treatment. D. A summary of MYC gene expression alteration following short-term exposure to CDK12 inhibitors. Note that robust MYC induction by CDK12 inhibition was also observed in the primary cultures of HER2+ metastatic breast tumor DFBC-1407

Article Snippet: The CDK12 inhibitor SR-4835 (TargetMol, #T8325) was dissolved in DMSO and aliquoted; upon use, the SR-4835 solution was mixed at a ratio of 1:9 with 30% HP-β-CD.

Techniques: Transformation Assay, Control, Western Blot, Expressing, Gene Expression, Inhibition

Journal: bioRxiv

Article Title: Mitotic phosphorylation of ADAR1 regulates its centromeric localization and is required for faithful mitotic progression

doi: 10.1101/2025.05.28.656747

Figure Lengend Snippet: (A) HeLa cells were collected under asynchronous (Async), mitotically arrested (M; nocodazole-treated), or S phase-arrested (S; thymidine-treated) conditions. Cell lysates were analyzed by SDS-PAGE with (+) or without (–) Phos-tag acrylamide to detect phosphorylated ADAR1p110. λ-Phosphatase treatment was used to confirm phosphorylation dependency. In Phos-tag gels (top panel), ADAR1p110 exhibited a mobility shift that was strongly enhanced in mitotic samples, appearing as multiple slower-migrating bands. This shift was abolished by phosphatase treatment, indicating that the observed shift is phosphorylation-dependent. Conventional SDS-PAGE (bottom panel) was performed to assess total ADAR1p110 and ADAR1p150 protein levels as loading controls. (B) Mass spectrometry-based phosphopeptide mapping was performed on 3×Flag-tagged ADAR1p110 purified from 293T cells under mitotically synchronized conditions. The amino acid sequence starting from residue 514 is shown. Orange marks indicate phosphorylation sites. Below the sequence, a schematic representation of ADAR1p110 is provided, including the Z-DNA binding domain (green), the dsRBDs (blue), and the deaminase domain (red). (C) HeLa cells were transfected with siRNA targeting the 3′-untranslated region (3′-UTR) of ADAR1, followed by transfection with mCherry-tagged ADAR1p110 constructs. The constructs included WT, phospho-mimetic mutants (3×D and S614D), and phospho-deficient mutants (3×A and S614A). Cells were harvested 48 h after transfection, and total lysates were analyzed by western blotting using antibodies against mCherry (exogenous ADAR1p110), endogenous ADAR1p110, phospho-histone H3 (Ser10), and GAPDH. Phospho-histone H3 (S10) band intensity was used as a readout for mitotic accumulation under each condition. (D) HeLa cells were treated with kinase inhibitors under Async or Msync conditions. Cells were exposed to PLK1 inhibitors BI2536 and GSK461364, and CDK12/13 inhibitors SR-4835 and THZ531, across indicated concentrations. Whole-cell lysates were analyzed by Phos-tag SDS-PAGE followed by immunoblotting to detect phosphorylated ADAR1p110. Phosphorylated forms were visualized as slower-migrating bands. A decrease or disappearance of these bands indicates a loss of phosphorylation upon kinase inhibition. (E) HeLa cells were synchronized in Msync using nocodazole and transfected with either control siRNA (siNC1) or CDK13-targeting siRNA (siCDK13). Asynchronous cells were included for reference. Whole-cell lysates were subjected to Phos-tag SDS-PAGE followed by western blotting to assess the phosphorylation status of ADAR1p110. A reduction in the slower-migrating phosphorylated form of ADAR1p110 was observed upon CDK13 knockdown, confirming its role in mitotic phosphorylation.

Article Snippet: Nocodazole (0.1 μg/mL) was then added together with kinase inhibitors, including BI-2536 (PLK1 inhibitor, Selleck), GSK461364 (PLK1 inhibitor, MedChemExpress), SR-4835 (CDK12/13 inhibitor, Selleck), or THZ531 (CDK12/13 inhibitor, Selleck).

Techniques: SDS Page, Phospho-proteomics, Mobility Shift, Mass Spectrometry, Purification, Sequencing, Residue, Binding Assay, Transfection, Construct, Western Blot, Inhibition, Control, Knockdown

Journal: EMBO Reports

Article Title: DDB1 engagement defines the selectivity of S656 analogs for cyclin K degradation over CDK inhibition

doi: 10.1038/s44319-025-00448-y

Figure Lengend Snippet: ( A ) Inhibitory profile of S656, CR8, SR4835, HQ461, NCT02, THZ531 and SNS-032 compounds across 40 primary AML specimens in dose response assays. IC 50 values were determined after 6 days of incubation and normalized to DMSO control. ( B ) Dot plot summarizing the IC 50 values and medians (red) obtained in the panel of 40 primary AMLs. ( C ) Heatmap representation and hierarchical clustering of IC 50 values from ( A ). Correlation between S656 and the other molecules is indicated. Relative color scheme uses the minimum and maximum IC 50 values in each row. .

Article Snippet: SR4835 , MedChemExpress , HY-130250.

Techniques: Incubation, Control

Journal: EMBO Reports

Article Title: DDB1 engagement defines the selectivity of S656 analogs for cyclin K degradation over CDK inhibition

doi: 10.1038/s44319-025-00448-y

Figure Lengend Snippet: ( A ) Dose response experiment to determine IC 50 values of the indicated compounds in OCI-AML5 cells expressing shRNAs targeting CDK1 , 2 , 4 , 7 , 9 , cyclin K or a control region (4 days incubation, mean +/− SD, n = 3, biological replicates, t test, P value = 0.0011 (sh ctrl vs sh Cyclin K) in CR8, P value = 0.0029 (sh ctrl vs sh Cyclin K) in SR4835, P value = 0.0011 (sh ctrl vs sh Cyclin K) in HQ461, P value = 0.0025 (sh ctrl vs sh CDK9) in SNS-032, P value = 0.0038 (sh ctrl vs sh CDK1) in S656, P value = 0.0253 (sh ctrl vs sh CDK9) in S656 and P value = 0.0004 (sh ctrl vs sh Cyclin K) in S656). ( B ) Dot plot representation of the IC 50 values of all S656 analogs (black) and control molecules (colors) in OCI-AML5 cells along with their efficiency to degrade cyclin K eGFP (percentage of degradation after 3 h incubation with 10 μM of compounds, compared to DMSO). Highest tested dose is 10000 nM. ( C ) Dot plot representation of the ratio of IC 50 values in OCI-AML5 expressing shRNA control or targeting CUL4A , and the percentage of cyclin K eGFP degradation (3 h incubation with 10 μM of compounds, compared to DMSO) for all active (IC 50 < 10000 nM) S656 analogs (black) and control molecules (colors). ( D ) Working model in which molecules with high affinity for DDB1 behave as cyclin K degraders while modifications of the left-hand side of the molecule, lowering or abolishing the interaction with DDB1, induce cytotoxicity through CDK inhibition. See also Appendix Fig. S . .

Article Snippet: SR4835 , MedChemExpress , HY-130250.

Techniques: Expressing, Control, Incubation, shRNA, Inhibition