Journal: Nature Communications

Article Title: Transcriptional regulation of protein synthesis by mediator kinase represents a therapeutic vulnerability in MYC-driven medulloblastoma

doi: 10.1038/s41467-025-64937-3

Figure Lengend Snippet: a Log-fold change in gene dependency from CRISPR-Cas9 screens in MB lines with TP53 as a positive control. b Read counts of sgRNAs targeting CDK8. Each dot represents an individual sgRNA. A decrease in read counts after puromycin selection indicates that CDK8 is selected as an essential gene. c DepMap lineage plot of CDK8 DEMETER2 scores across cancer types. n = cell lines per lineage. Lower scores indicate greater dependency. Boxes = Q1, median, Q3; whiskers = 1.5× IQR. Statistics from DepMap. d MB-specific gene-dependency t-statistics from DepMap; values < 0 denote essential genes. e UMAP analysis of cell clusters of single-cell RNA-seq data from G3-MB patient samples. A total of 12,595 cells were plotted after quality-control filtering. f CDK8 immunofluorescence staining. Data points from n = 3 biological replicates. Scale bar, 100 μm. Line indicates median. One-way ANOVA. g Proliferation of shNull/shCDK8 D425 ( n = 4) and D458 ( n = 5) cells. Technical replicates. h Images of neurosphere size in CDK8 knockdown and control cells are shown. n = 5 technical replicates. Scale bar, 400 μm. Barplot is for day 10. i Sphere formation and self-renewal measured by ELDA in shNull/shCDK8 MB lines. P values: likelihood ratio test. n = 5 biological replicates . j CDK8 knockout reduces D458 proliferation. n = 3 technical replicates. k Representative images of neurospheres formed by CDK8 knockout ( n = 5) and control cells ( n = 5). Scale bar, 200 μm. l Immunofluorescence staining of CDK8 in xenograft tumors from shNull or shCDK8 D458. White dashed lines = tumor boundary. n = 2 (shNull), n = 3 (shCDK8); one shNull mouse died before scan. Scale bar, 100 μm. Box plot line = mean. Two-sided t-test. m Representative MRI of xenografts. n = 2 (shNull), n = 3 (shCDK8). n H&E shows reduced tumor formation. Three mice per group euthanized at day 20. o Kaplan–Meier survival analysis of shNull ( n = 6) and shCDK8 ( n = 5) mice. Log-rank test.

Article Snippet: The following antibodies were used: phospho-4EBP1 (Santa Cruz Biotechnology, sc-293124, 1:50), CDK8 (Santa Cruz Biotechnology, sc-13155, 1:50), CDK8 (Abcam, ab224828, 1:200), CDK8 (Invitrogen, PA5-11500, 1:200), fibrillarin (Abcam, EPR10823 , 1:200), nucleolin (Abcam, EPR7952, 1:200), and ribosomal RNA antibody Y10B (Abcam, ab171119, 1:200) for 1 h at room temperature.

Techniques: CRISPR, Positive Control, Selection, RNA Sequencing, Control, Immunofluorescence, Staining, Knockdown, Knock-Out

Journal: Nature Communications

Article Title: Transcriptional regulation of protein synthesis by mediator kinase represents a therapeutic vulnerability in MYC-driven medulloblastoma

doi: 10.1038/s41467-025-64937-3

Figure Lengend Snippet: a IC50 determination of various CDK8 inhibitors in MB cell lines. Unit: μmol. b IC50 of RVU120 at 72 h in MB and NHA cells. Experiments were performed in n = 3 independent experiments. All comparisons are to NHA cells. One-way ANOVA. c Dose-dependent proliferation curve of RVU120-treated primary MB cells from a G3-MB patient. n = 5 biological replicates. Mea n ± SEM. One-way ANOVA. d Immunofluorescence of CDK8 and DAPI. MB cells were treated with 1000 nM RVU120 for 48 h. Data points from n = 3 biological replicates. Scale bar, 10 μm. The line on the box plot represents the median. Two-sided unpaired t-test. e Immunoblot analysis of p-STAT1 following time-dependent treatment with 1000 nM RVU120 across MB cell lines. Representative of n = 3 experiments. f Immunoblot analysis of p-STAT1 levels following dose-dependent RVU120 treatment at 72 h. Representative of n = 3 experiments. Mea n ± SEM. Two-way ANOVA. g Methylcellulose assay in MB cells treated with RVU120. n = 3 biological replicates. Mea n ± SEM. Two-way ANOVA. h Annexin V apoptosis assay. MB cells were treated with 1000 nM RVU120 for 48 h. n = 3 biological replicates. Mea n ± SEM. Two-way ANOVA. i Identification of the brain tumor-initiating cell fraction in MB cells by ALDH expression demonstrates a decrease in the ALDH + fraction following 1000 nM RVU120 treatment for 48 h. n = 3 biological replicates. Mea n ± SEM. Two-way ANOVA. j Representative bioluminescence images of mice treated with RVU120 or vehicle (control, n = 8; RVU120, n = 6). k Kaplan–Meier survival curves of mice treated with vehicle ( n = 8) or RVU120 ( n = 6). Log-rank test. l Representative MRI of PDX411 xenograft mice treated with RVU120 or vehicle. Mice received the first scan after 14 days of treatment. Asterisks denote spongy tissue texture in mice. An adjusted texture analysis was performed to measure the tumor size. n = 3 mice. One control mouse died before the final scan. Mea n ± SEM. Two-way ANOVA.

Article Snippet: The following antibodies were used: phospho-4EBP1 (Santa Cruz Biotechnology, sc-293124, 1:50), CDK8 (Santa Cruz Biotechnology, sc-13155, 1:50), CDK8 (Abcam, ab224828, 1:200), CDK8 (Invitrogen, PA5-11500, 1:200), fibrillarin (Abcam, EPR10823 , 1:200), nucleolin (Abcam, EPR7952, 1:200), and ribosomal RNA antibody Y10B (Abcam, ab171119, 1:200) for 1 h at room temperature.

Techniques: Immunofluorescence, Western Blot, Methylcellulose Assay, Apoptosis Assay, Expressing, Control

Journal: Nature Communications

Article Title: Transcriptional regulation of protein synthesis by mediator kinase represents a therapeutic vulnerability in MYC-driven medulloblastoma

doi: 10.1038/s41467-025-64937-3

Figure Lengend Snippet: a DEMETER2 scores reveal differing CDK8/19 dependency in high- vs. low-MYC medulloblastoma cells. Two-sides Mann–Whiteny test. b Microarray analysis of CDK8 and CDK19 expression across four subgroups and twelve subtypes of 763 MB patient samples (Cavalli et al.) n = 6 normal cerebellum samples were collected by us. Boxes represent the first, median, and third quartiles, with whiskers extending to 1.5× the interquartile range. Two-sided Wilcoxon test. The p-values for the subtypes are provided in the supplementary information. c Kaplan–Meier survival analysis showing the association between CDK8 expression and overall survival within Group 3 subtypes. Log-rank test. Group 3β: n = 10 high, 17 low; Group 3r: n = 18 high, 13 low; Group 3β + r: n = 50 high, 8 low. d Correlation analysis of CDK8 and CDK19 with MYC or MYCN expression in Group 3 and SHH subgroups; IMPDH2 and MYCNOS were included as positive controls. e IGV tracks showing c-MYC ChIP-seq peaks at the promoters of CDK8 and CDK19; IMPDH2 and ODC1 serve as positive controls with MYC binding peaks. f RNA-seq analysis following knockdown of c-MYC shows no change in CDK8 expression in MB002 cells. n = 2 for each condition. g RNA-seq analysis following CDK8 knockdown with three shRNAs, showing the expression levels of both c-MYC and CDK8 in D458 cells. n = 3 for each condition. P-values were adjusted using the Benjamini–Hochberg FDR and reported as padj (DESeq2). h Volcano plot showing differentially expressed genes in CDK8 knockdown cells compared to shNull control cells. P-values were adjusted using the Benjamini–Hochberg FDR and reported as padj (DESeq2). i Immunoblot showing CDK8 and c-MYC protein levels in MB cells following CDK8 knockdown. n = 3 independent experiments. Mea n ± SEM. Two-way ANOVA. j Immunoblot showing CDK8 and c-MYC protein levels in MB cells with CRISPR/Cas9-mediated knockout of CDK8. n = 3 independent experiments. Mean ± SEM. Two-way ANOVA. k Gene set enrichment analysis of hallmark gene sets using RNA-seq data comparing CDK8 knockdown (shCDK8) to control (shNull) cells. n = 3 for each condition.

Article Snippet: The following antibodies were used: phospho-4EBP1 (Santa Cruz Biotechnology, sc-293124, 1:50), CDK8 (Santa Cruz Biotechnology, sc-13155, 1:50), CDK8 (Abcam, ab224828, 1:200), CDK8 (Invitrogen, PA5-11500, 1:200), fibrillarin (Abcam, EPR10823 , 1:200), nucleolin (Abcam, EPR7952, 1:200), and ribosomal RNA antibody Y10B (Abcam, ab171119, 1:200) for 1 h at room temperature.

Techniques: Microarray, Expressing, ChIP-sequencing, Binding Assay, RNA Sequencing, Knockdown, Control, Western Blot, CRISPR, Knock-Out

Journal: Nature Communications

Article Title: Transcriptional regulation of protein synthesis by mediator kinase represents a therapeutic vulnerability in MYC-driven medulloblastoma

doi: 10.1038/s41467-025-64937-3

Figure Lengend Snippet: a Alterations in GSEA gene sets observed in D458 cells following CDK8 knockdown. FDR from GSEA. b GSVA of patient samples ( n = 763) showed enrichment of ribosome biogenesis gene sets in MYC-overexpressing Group 3β/3γ subtypes. c The expression of ribosomal genes was compared to that of all other genes in MB cells using RNA-seq analysis. d RNA-seq analysis demonstrated alterations in the expression of mitochondrial and cytoplasmic ribosomal genes following loss or inhibition of CDK8 in MB cells. n = 3 for each condition. P-values were FDR-adjusted. e RNA-seq of D458 cells: cytosolic ribosomal gene expression in shCDK8, sgCDK8, and control. n = 3 for each condition. f GSEA network showing downregulation of ribosome biogenesis in shCDK8 vs. shNull D458 cells. Node size reflects gene counts; edges indicate shared genes. g Top 10 GO biological processes are shown ( n = 3 per condition; FDR-adjusted P values, GSEA). h Polysome profiling of lysates from CDK8 knockout or RVU120-treated (1 μM, 48 h) D458 cells reveals that CDK8 depletion reduces the ratio of polysome to sub-polysome compared to controls. i Immunofluorescence of Y10B and DAPI at 40X. MB cells were treated with the 1000 nM RVU120 for 48 h. Scale bar, 10 μm. Median line. n = 3 independent experiments. Two-sided t-test. j Immunofluorescence staining shows EU-incorporated RNA detected by Click-iT labeling. Scale bar, 100 μm. The line on the box plot represents the mean. n = 3 independent experiments. Kruskal-Wallis test. k CRISPR-mediated CDK8 knockout and control D458 cells were labeled with EU for 1 hour, and detected using the Click-iT reaction. Scale bar, 100 μm. Line indicates mean. n = 3 independent experiments. Two-sided Mann-Whitney test. l Flow cytometry analysis of OPP incorporation in MB cells treated with RVU120 at 1,000 nM versus control, quantified using FlowSight. Scale bar, 10 μm. Mean ± SEM. n = 3 independent experiments. Two-way ANOVA. m OPP assay showing protein synthesis in RVU120-treated MB cells compared to control cells. Scale bar, 100 μm. Line indicates mean. n = 3 independent experiments. Kruskal-Wallis test.

Article Snippet: The following antibodies were used: phospho-4EBP1 (Santa Cruz Biotechnology, sc-293124, 1:50), CDK8 (Santa Cruz Biotechnology, sc-13155, 1:50), CDK8 (Abcam, ab224828, 1:200), CDK8 (Invitrogen, PA5-11500, 1:200), fibrillarin (Abcam, EPR10823 , 1:200), nucleolin (Abcam, EPR7952, 1:200), and ribosomal RNA antibody Y10B (Abcam, ab171119, 1:200) for 1 h at room temperature.

Techniques: Knockdown, Expressing, RNA Sequencing, Inhibition, Gene Expression, Control, Knock-Out, Immunofluorescence, Staining, Labeling, CRISPR, MANN-WHITNEY, Flow Cytometry

Journal: Nature Communications

Article Title: Transcriptional regulation of protein synthesis by mediator kinase represents a therapeutic vulnerability in MYC-driven medulloblastoma

doi: 10.1038/s41467-025-64937-3

Figure Lengend Snippet: a Top 10 genes showing the highest correlation with MYC expression in Group 3 MB, based on analysis of the Cavalli dataset. n = 763. b GSEA of C5 GO gene sets comparing shMYC versus shNull in MB cells. P-values were adjusted for multiple testing using the Benjamini–Hochberg FDR in GSEA. n = 2 for each condition. c GSEA indicated alterations in GO biological process gene sets (FDR < 0.05) following the knockdown of CDK8, MYC, CDK7, CDK11, HNRNPH1, SOX11, or PLK1. n = 3 for each condition. d OPP assay in D458 and MB002 cells transfected with GFP-shRNA targeting MYC, followed by treatment with two doses of RVU120. Scale bar, 100 μm. The line on the box plot represents the median. n = 3 independent experiments. Kruskal-Wallis test. e OPP assay in D458 cells transfected with RFP-Omomyc, where RFP-positive cells mark Omomyc expression. Scale bar, 100 μm. The line on the box plot represents the median. n = 3 independent experiments. Two-sided Mann-Whitney test. f OPP assay in D458 cells transfected with RFP-Omomyc, followed by treatment with 1,000 nM RVU120. White arrows mark Omomyc-expressing (RFP-positive) cells. Scale bar, 100 μm. The line on the box plot represents the median. n = 3 independent experiments. Kruskal-Wallis test. g OPP assay showing dose-dependent effects of RVU120 treatment on ONS76 cells. Scale bar, 100 μm. The line on the box plot represents the median. n = 3 independent experiments. Kruskal-Wallis test. h OPP assay showing dose-dependent effects of RVU120 treatment on ONS76 cells transfected with c-MYC. Scale bar, 100 μm. The line on the box plot represents the median. n = 3 independent experiments. Kruskal-Wallis test. i IC₅₀ of RVU120 in parental ONS76 and DAOY cells compared to c-MYC–overexpressing cells. Mean ± SD. Two-sided unpaired t-test. Experiments were performed in three independent experiments, each with five biological replicates.

Article Snippet: The following antibodies were used: phospho-4EBP1 (Santa Cruz Biotechnology, sc-293124, 1:50), CDK8 (Santa Cruz Biotechnology, sc-13155, 1:50), CDK8 (Abcam, ab224828, 1:200), CDK8 (Invitrogen, PA5-11500, 1:200), fibrillarin (Abcam, EPR10823 , 1:200), nucleolin (Abcam, EPR7952, 1:200), and ribosomal RNA antibody Y10B (Abcam, ab171119, 1:200) for 1 h at room temperature.

Techniques: Expressing, Knockdown, Transfection, shRNA, MANN-WHITNEY

Journal: Nature Communications

Article Title: Transcriptional regulation of protein synthesis by mediator kinase represents a therapeutic vulnerability in MYC-driven medulloblastoma

doi: 10.1038/s41467-025-64937-3

Figure Lengend Snippet: a Heatmaps showing CUT&RUN signals of CDK8, H3K4me3, H3K4me1, H3K27ac, BRD4, and MYC in D458 MB cells. The signals were displayed within a region spanning ± 3 kb around the transcription start site (TSS). n = 2 for each condition. b Pie chart showing CDK8 peaks are localized at promoter and enhancer. n = 2 for each condition. c Pathway enrichment analysis of CDK8 binding genes inferred from CUT&RUN. Translation pathways are enriched in MB cell lines. A total of 11,675, 14,909, and 12,895 genes were identified in D458, D425, and D283 cells, respectively. Statistical significance was assessed using Fisher’s exact test with the total number of genes in the genome as the background, and p-values were adjusted for multiple testing using the Benjamini–Hochberg FDR. d Venn-diagram showing overlapping of CDK8 binding genes associated with mRNA translation pathways. e Heatmaps displaying genome-wide binding CUT&RUN signals of CDK8 in CDK8 knockdown D458 cells compared to control cells. The signals are displayed within a region spanning ± 3 kb around the transcription start site (TSS). n = 2 for each condition. f Heatmaps displaying CUT&RUN signals of CDK8 and H3K4me3 in D458 cells with CDK8 knockdown compared to control cells at promoter regions. n = 2 for each condition. g Pathway enrichment analysis of genes associated with loss of H3K4me3 peaks (1207 genes). Statistical significance was assessed using Fisher’s exact test with the total number of genes in the genome as the background, and p-values were adjusted for multiple testing using the Benjamini–Hochberg FDR. h Heatmaps showing CUT&RUN signals of BRD4, H3K4me1, and MYC in D458 MB cells following CDK8 knockdown. n = 2 for each condition.

Article Snippet: The following antibodies were used: phospho-4EBP1 (Santa Cruz Biotechnology, sc-293124, 1:50), CDK8 (Santa Cruz Biotechnology, sc-13155, 1:50), CDK8 (Abcam, ab224828, 1:200), CDK8 (Invitrogen, PA5-11500, 1:200), fibrillarin (Abcam, EPR10823 , 1:200), nucleolin (Abcam, EPR7952, 1:200), and ribosomal RNA antibody Y10B (Abcam, ab171119, 1:200) for 1 h at room temperature.

Techniques: Binding Assay, Genome Wide, Knockdown, Control

Journal: Nature Communications

Article Title: Transcriptional regulation of protein synthesis by mediator kinase represents a therapeutic vulnerability in MYC-driven medulloblastoma

doi: 10.1038/s41467-025-64937-3

Figure Lengend Snippet: a Heatmaps showing CUT&RUN signals of Pol II and phospho-Pol II in D458 cells with CDK8 knockdown compared to control cells at promoter regions. n = 2 for each condition. b Empirical cumulative distribution function (ECDF) plot shows significant increase in promoter-proximal pausing following CDK8 knockdown. n = 2 for each condition. c Average distribution and heatmaps of H3K4me3, Pol II, and phospho-Pol II signals on ribosomal genes. n = 2 for each condition. d Representative examples of Pol II and phospho-Pol II binding sites on ribosomal genes observed following CDK8 knockdown. n = 2 for each condition. e Enrichment analysis shows mRNA translation pathways enriched among genes with increased Pol II peaks (11,617 genes) or decreased phospho-Pol II peaks (7174 genes) following CDK8 knockdown. Statistical significance was assessed using Fisher’s exact test with the total number of genes in the genome as the background, and p-values were adjusted for multiple testing using the Benjamini–Hochberg FDR. f Immunoblot showing the levels of Pol II and phospho-Pol II in D458 cells following treatment with RVU120. Representative of n = 3 experiments. g Heatmaps showing CUT&RUN signals of RNA Pol II and phospho-RNA Pol II in D458 MB cells treated with 1,000 nM RVU120 for 48 h. n = 2 for each condition. h Average distribution of RNA Pol II and phospho-RNA Pol II peaks showing the alteration of RNA Pol II and phospho-RNA Pol II signals across the gene body following the treatment of RVU120. n = 2. i Average distribution and heatmaps of RNA Pol II and phospho-RNA Pol II signals on cytosolic and mitochondrial ribosomal genes following the treatment of RVU120. n = 2. j Representative examples of RNA Pol II and phospho-RNA Pol II binding sites on ribosomal genes observed following the treatment of RVU120. n = 2 for each condition.

Article Snippet: The following antibodies were used: phospho-4EBP1 (Santa Cruz Biotechnology, sc-293124, 1:50), CDK8 (Santa Cruz Biotechnology, sc-13155, 1:50), CDK8 (Abcam, ab224828, 1:200), CDK8 (Invitrogen, PA5-11500, 1:200), fibrillarin (Abcam, EPR10823 , 1:200), nucleolin (Abcam, EPR7952, 1:200), and ribosomal RNA antibody Y10B (Abcam, ab171119, 1:200) for 1 h at room temperature.

Techniques: Knockdown, Control, Binding Assay, Western Blot

Journal: Nature Communications

Article Title: Transcriptional regulation of protein synthesis by mediator kinase represents a therapeutic vulnerability in MYC-driven medulloblastoma

doi: 10.1038/s41467-025-64937-3

Figure Lengend Snippet: a Gene set variation analysis of patient samples ( n = 763) revealed that the MYC-overexpressing subtypes Group3β and 3γ were enriched with gene sets of MYC and mTOR signaling. b Multiplex IHC on G3-MB patient samples using CDK8, p-4EBP1, c-MYC, RPS12, p-S6, and p-AKT antibodies. n = 3 patient samples were analyzed. For each antibody, 10 fields of view were captured, and the signal intensities from all points were combined for analysis. Top scale bar, 100 μm. Bottom scale bar, 10 μm. p < 0.0001 in all biomarker groups. Two-sided Mann–Whitney U. c Representative bioluminescence images of mice injected with D425 cells and treated with TAK-228 (1 mg/kg, daily, oral gavage) or vehicle control. Treatment was initiated upon tumor establishment and continued until endpoint. Kaplan–Meier survival curves show extended survival in the TAK-228–treated cohort compared to controls. Statistical significance was determined using the log-rank test. n = 5 for each group. d Representative bioluminescence images and Kaplan–Meier survival analysis of D458 xenograft-bearing mice treated with TAK-228 (1 mg/kg, daily, oral gavage), following the same protocol as in ( c ). TAK-228 treatment reduced tumor burden and significantly prolonged survival compared to the control group (log-rank test). n = 5 for each group. e GSEA plots of representative gene sets involved in mTOR signaling following CDK8 depletion or inhibition. Normalized enrichment score (NES) and false discovery rate (FDR) are indicated. n = 3 for each condition. f –h Immunoblot showing the levels of p-4EBP1 and p-S6 upon treatment with RVU120 in Group 3 medulloblastoma cells. n = 3 biological replicates. i Immunoblot showing the levels of p-4EBP1 and p-S6 following CDK8 knockout. n = 3 independent experiments. Mea n ± SEM. Statistical analysis: two-way ANOVA. j Immunoblot showing the levels of p-mTOR and mTOR upon treatment with RVU120 for 24 h in D458 and MB002 cells. The data are representative of n = 3 independent experiments.

Article Snippet: The following antibodies were used: phospho-4EBP1 (Santa Cruz Biotechnology, sc-293124, 1:50), CDK8 (Santa Cruz Biotechnology, sc-13155, 1:50), CDK8 (Abcam, ab224828, 1:200), CDK8 (Invitrogen, PA5-11500, 1:200), fibrillarin (Abcam, EPR10823 , 1:200), nucleolin (Abcam, EPR7952, 1:200), and ribosomal RNA antibody Y10B (Abcam, ab171119, 1:200) for 1 h at room temperature.

Techniques: Multiplex Assay, Biomarker Discovery, MANN-WHITNEY, Injection, Control, Inhibition, Western Blot, Knock-Out

Journal: Science immunology

Article Title: Conversion of antigen-specific effector/memory T cells into Foxp3-expressing T reg cells by inhibition of CDK8/19.

doi: 10.1126/sciimmunol.aaw2707

Figure Lengend Snippet: Fig. 3. Interaction of CDK8/19 and STAT5 in inducing Foxp3 expression. (A) Mouse CD4+ T cells were mock-infected or infected with retrovirus harboring the WT CDK8 and STAT5b genes, stimulated with anti-CD3/CD28 and IL-2, and subjected to immunoprecipitation and immunoblotting for CDK8, STAT5b, and MED12. Data are representative of two independent experiments. (B) STAT5 serine phosphorylation by CDK8. Recombinant GST-STAT5b incubated with recombinant WT or KD CDK8 in the presence or absence of 1.0 M AS with 100 M ATP and 10 mM MgCl2 was subjected to immunoblotting for phosphoserine (pS) of STAT5b. Data are representative of two independent experiments. (C) Control of serine and tyrosine phos- phorylation by AS in activated T cells. Mouse CD4+ T cells were stimulated with anti-CD3/CD28 in the presence or absence of TGF- for 22 hours and in the absence (DMSO) or presence of 100 nM AS, lysed, and subjected to immunoblot analysis for STAT5b, pS-STAT5b, or pY-STAT5. Signal intensity was quantified and normalized by GAPDH (n = 3 or 4). **P < 0.01 (Student’s t test). AU, arbitrary units. (D and E) Mouse naїve CD4+ T cells were incubated in the presence or absence of anti-CD3/28 for 22 hours, and PLA was performed to assess interaction between CDK8 and STAT5. Images were obtained using an LSM710 confocal microscope. Data were presented as maximum intensity projection (n = 3). Each red spot represents a single interaction, and DNA was stained with DAPI. (F) Mouse naїve CD4+ T cells were incubated in the presence or absence of anti-CD3/CD28 for 22 hours and examined for expression of CDK8 and STAT5. DNA was stained with Hoechst33342. Images were obtained using an LSM710 confocal microscope. Data are representative of two independent experiments. (G) Mouse CD4+ T cells infected with retrovirus encoding WT or S730A mutant STAT5b were stimulated with anti-CD3/CD28 and IL-2, without TGF-, and subjected to immunoblotting for STAT5b (left), or assessed for the percentage of Foxp3+ cells among live virus–infected (i.e., GFP+) CD4+ T cells by flow cytometry (n = 7) (right). ***P < 0.001 (Student’s t test).

Article Snippet: A fragment encoding mouse CDK8, CDK19, or STAT5b cDNA was obtained from pCMV6-CDK8 (OriGene, MR218219), pCMV6-CDK19 (OriGene, MR215712), or pCMV6-STAT5b (OriGene, MR210649) and subcloned into a retroviral plasmid pMCs-IRES-GFP (Cell Biolabs).

Techniques: Expressing, Infection, Immunoprecipitation, Western Blot, Phospho-proteomics, Recombinant, Incubation, Control, Microscopy, Staining, Mutagenesis, Virus, Flow Cytometry

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

Journal: International Journal of Molecular Sciences

Article Title: CDK8 Inhibition Increases E2F1 Transcriptional Activity and Promotes STAT3-Dependent Suppression of Mcl-1 in Triple-Negative Breast Cancer Cell Line MDA-MB-468

doi: 10.3390/ijms27020897

Figure Lengend Snippet: Effect of CDK8 inhibitor Q12 treatment of MDA-MB-468 cells on E2F1 protein and E2F1-dependent luciferase expression. ( A ) Effect of treatment with 10 µM Q12 (6 h and 24 h) on E2F1 ( left ) and pE2F1(Ser375) ( right ) expression in MDA-MB-468 cells compared to vehicle-treated control (DMSO) cells ( n = 3–6). ( B ) Relative luciferase expression in MDA-MB-468 cells transfected with an E2F1-responsive luciferase plasmid following treatment with Q12 (10 µM) or SEL120 (1µM) compared to vehicle-treated control (DMSO) cells ( n = 4). Unpaired Student’s t -test was used to determine significance. * ρ < 0.05, ** ρ < 0.01, **** ρ < 0.0001.

Article Snippet: Control siRNA-B (sc-44230), E2F-1 siRNA (h) (sc-29297), Cdk8 siRNA (h) (sc-29267), siRNA Transfection Medium (sc-36868) and siRNA Transfection Reagent (sc-29528), and mouse anti-Mcl-1 (22, sc-12756) (dil. 1:400) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Luciferase, Expressing, Control, Transfection, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: CDK8 Inhibition Increases E2F1 Transcriptional Activity and Promotes STAT3-Dependent Suppression of Mcl-1 in Triple-Negative Breast Cancer Cell Line MDA-MB-468

doi: 10.3390/ijms27020897

Figure Lengend Snippet: ( A ) p73 protein expression following treatment with 10 µM Q12 (1, 6, 12, 24 h) compared to vehicle-treated (DMSO) control ( n = 3). ( B ) CDK8 protein expression in MDA-MB-468 cell line following 24 h treatment with 10 µM Q12 ( n = 3). Images are representative of three independent experiments. Error bars represent mean ± SD. Unpaired Student’s t -test was used to determine significance. ** ρ < 0.01.

Article Snippet: Control siRNA-B (sc-44230), E2F-1 siRNA (h) (sc-29297), Cdk8 siRNA (h) (sc-29267), siRNA Transfection Medium (sc-36868) and siRNA Transfection Reagent (sc-29528), and mouse anti-Mcl-1 (22, sc-12756) (dil. 1:400) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: CDK8 Inhibition Increases E2F1 Transcriptional Activity and Promotes STAT3-Dependent Suppression of Mcl-1 in Triple-Negative Breast Cancer Cell Line MDA-MB-468

doi: 10.3390/ijms27020897

Figure Lengend Snippet: ( A ) Western blot assessment of siRNA-mediated CDK8 KD. ( B ) Western blot assessment of siRNA-mediated E2F1 KD. ( C ) Comparison of cell viability of wild-type, siRNA control-transfected, CDK8 siRNA-transfected, and E2F1 siRNA-transfected MDA-MB-468 cells treated with Q12 (20 µM) for 0.5, 1, 2, 6, and 24 h ( n = 3). Only significant differences are shown. * ρ ≤ 0.01.

Article Snippet: Control siRNA-B (sc-44230), E2F-1 siRNA (h) (sc-29297), Cdk8 siRNA (h) (sc-29267), siRNA Transfection Medium (sc-36868) and siRNA Transfection Reagent (sc-29528), and mouse anti-Mcl-1 (22, sc-12756) (dil. 1:400) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Western Blot, Comparison, Control, Transfection

Journal: International Journal of Molecular Sciences

Article Title: CDK8 Inhibition Increases E2F1 Transcriptional Activity and Promotes STAT3-Dependent Suppression of Mcl-1 in Triple-Negative Breast Cancer Cell Line MDA-MB-468

doi: 10.3390/ijms27020897

Figure Lengend Snippet: ( A ) Expression of Mcl-1 protein in MDA-MB-468 cells treated with Q12 (10 µM, 24 h), cryptotanshinone (CPT, 10 µM, 24 h), or co-treatment with Q12 (10 µM, 24 h) and CPT (10 µM, 24 h) compared to vehicle-treated (DMSO) control cells ( n = 3). ( B ) Proposed mechanism by which CDK8 inhibitor decreases viability of MDA-MB-468 triple-negative breast cancer cells. Images are representative of three independent experiments. Error bars represent mean ± SD. Unpaired Student’s t -test was used to determine significance. * ρ < 0.05, *** ρ < 0.001, ns = not significant.

Article Snippet: Control siRNA-B (sc-44230), E2F-1 siRNA (h) (sc-29297), Cdk8 siRNA (h) (sc-29267), siRNA Transfection Medium (sc-36868) and siRNA Transfection Reagent (sc-29528), and mouse anti-Mcl-1 (22, sc-12756) (dil. 1:400) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Control