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Cell Signaling Technology Inc cdk8 antibody

A. Volcano plot of ssGSEA on genome-wide differential effect size of CORUM complexes comparing aRMS to other non-RMS tumor cell lines. Red indicates Mediator complex. B. Distribution of <t>CDK8</t> gene effect score across different cancer cell lines from the Broad Institute’s CRISPR Dependency Map (24Q2). C. Dot plot of kinase dependencies in the Broad Institute’s CRISPR Dependency Map comparing fusion-positive RMS to all other cancer cell lines. CDK8 is highlighted in red. D. Violin plots showing distribution of CCNC , MED13 , and MED12 gene effect score from the Broad Institute’s CRISPR Dependency Map (24Q2) comparing the fusion-positive aRMS and fusion-negative eRMS with all other indicated cancer cell lines. aRMS is highlighted in red and eRMS is highlighted in blue. E. shRNA-mediated suppression of CDK8 by two different shRNAs impairs Rh30 and Rh28 aRMS cell growth in vitro . Cell numbers were determined by trypan blue live cell counting. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). F. Line graph showing mean subcutaneous tumor volume (mm3) formed by Rh28 cells after treatment with inducible knock down of CDK8 using shRNA. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). G. CRISPR-mediated knockout of CDK8 by two different gRNAs impairs Rh30 and Rh4 aRMS cell growth in vitro . Relative growth was assessed by CellTiter-Glo after CRISPR knockout. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04).

Cdk8 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma"

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

Journal: bioRxiv

doi: 10.1101/2025.07.14.663986

Figure Legend Snippet: A. Volcano plot of ssGSEA on genome-wide differential effect size of CORUM complexes comparing aRMS to other non-RMS tumor cell lines. Red indicates Mediator complex. B. Distribution of CDK8 gene effect score across different cancer cell lines from the Broad Institute’s CRISPR Dependency Map (24Q2). C. Dot plot of kinase dependencies in the Broad Institute’s CRISPR Dependency Map comparing fusion-positive RMS to all other cancer cell lines. CDK8 is highlighted in red. D. Violin plots showing distribution of CCNC , MED13 , and MED12 gene effect score from the Broad Institute’s CRISPR Dependency Map (24Q2) comparing the fusion-positive aRMS and fusion-negative eRMS with all other indicated cancer cell lines. aRMS is highlighted in red and eRMS is highlighted in blue. E. shRNA-mediated suppression of CDK8 by two different shRNAs impairs Rh30 and Rh28 aRMS cell growth in vitro . Cell numbers were determined by trypan blue live cell counting. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). F. Line graph showing mean subcutaneous tumor volume (mm3) formed by Rh28 cells after treatment with inducible knock down of CDK8 using shRNA. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). G. CRISPR-mediated knockout of CDK8 by two different gRNAs impairs Rh30 and Rh4 aRMS cell growth in vitro . Relative growth was assessed by CellTiter-Glo after CRISPR knockout. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04).

Techniques Used: Genome Wide, CRISPR, shRNA, In Vitro, Cell Counting, Knockdown, Knock-Out

A. Correlation between CDK8 gene dependency and sensitivity to the CDK8 inhibitor BI-1347 in 669 cancer cell lines (y-axis showing CDK8 dependency score; x-axis showing PRISM LFC value of BI-1347 treatment). Linear regression lines and Pearson correlation coefficients (R) are shown for all cell lines (black), aRMS (red), and eRMS (blue). B. Western blot analysis showing CDK8 inhibition by small molecules as assessed by STAT1 phosphorylation at serine 727. C, D. Dose response curves of inactive BI-1347 analog (BI-1374) (C) and three pharmacologic CDK8 inhibitors (D), BI-1347, SEL-120-34A, and JH-XII-178, at day 7 of treatment in Rh30, Rh4 and RHJT cell lines. E, F. Live cell proliferation assessed by Incucyte for Rh30 (E) and Rh4 (F) cells after treatment with vehicle DMSO (black), the CDK8 inhibitor BI-1347 (red) and its inactive analog BI-1374 (gray). Data were normalized to DMSO. Data represent means ± SEM (n=6, *: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). G. Line graph reveals mean subcutaneous tumor volume (mm ) formed by Rh30 cells after treatment with the CDK8 inhibitor SEL-120-34A. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04).

Figure Legend Snippet: A. Correlation between CDK8 gene dependency and sensitivity to the CDK8 inhibitor BI-1347 in 669 cancer cell lines (y-axis showing CDK8 dependency score; x-axis showing PRISM LFC value of BI-1347 treatment). Linear regression lines and Pearson correlation coefficients (R) are shown for all cell lines (black), aRMS (red), and eRMS (blue). B. Western blot analysis showing CDK8 inhibition by small molecules as assessed by STAT1 phosphorylation at serine 727. C, D. Dose response curves of inactive BI-1347 analog (BI-1374) (C) and three pharmacologic CDK8 inhibitors (D), BI-1347, SEL-120-34A, and JH-XII-178, at day 7 of treatment in Rh30, Rh4 and RHJT cell lines. E, F. Live cell proliferation assessed by Incucyte for Rh30 (E) and Rh4 (F) cells after treatment with vehicle DMSO (black), the CDK8 inhibitor BI-1347 (red) and its inactive analog BI-1374 (gray). Data were normalized to DMSO. Data represent means ± SEM (n=6, *: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). G. Line graph reveals mean subcutaneous tumor volume (mm ) formed by Rh30 cells after treatment with the CDK8 inhibitor SEL-120-34A. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04).

Techniques Used: Western Blot, Inhibition, Phospho-proteomics

A. Volcano plots showing the number of gene body changes after 24 hrs of CDK8 inhibitor BI-1347 treatment. Significantly up-regulated genes are highlighted in red; significantly downregulated genes are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5). B. Metagene plots of PRO-seq reads of genes with significant upregulation or downregulation of gene body transcription at 24 hrs of BI-1347 treatment. C. Heatmap of Log 2 transformed fold change values of PRO-seq pausing indices of genes with changes in gene body transcription at the 24 hr time point. D. Heatmap showing log 2 transformed fold change of RNA-seq read counts for indicated gene sets after DMSO and BI-1347 (10 nM) treatment for 24 or 72 hrs. E. Bubble dot plot shows Gene Set Enrichment Analysis (GSEA) of PRO-seq gene hits that are upregulated at 24 hrs. Dot size indicates number of genes in each gene set, and dot color indicates p -adjusted value. F. Immunofluorescence analysis of myogenin (green; 20X) in Rh30 (left) and Rh4 (right) cells after 7 days of DMSO or BI-1347 treatment. G. Representative images of H&E stain of xenograft tumors. Arrows indicate myofibrils. H. Volcano plots reveal the number of changes in enhancer RNA (eRNA) transcripts after 24 hrs of BI-1347 treatment. Significantly upregulated eRNAs are highlighted in red; significantly downregulated eRNAs are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5) I. MA plots showing the changes of chromatin accessibility by ATAC-seq following 4 and 24 hrs of BI-1347 treatment. Significantly increased ATAC-seq peaks are highlighted in red; significantly decreased ATAC-seq peaks are highlighted in black (padj<0.05, fold change>1.5 or <-1.5). J. Pie chart showing the peak annotation of up-regulated ATAC-seq peaks to gene features at 24 hrs of BI-1347 treatment. K. Histogram of PRO-seq reads around up-regulated ATAC-seq peaks at indicated time points of BI-1347 treatment.

Figure Legend Snippet: A. Volcano plots showing the number of gene body changes after 24 hrs of CDK8 inhibitor BI-1347 treatment. Significantly up-regulated genes are highlighted in red; significantly downregulated genes are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5). B. Metagene plots of PRO-seq reads of genes with significant upregulation or downregulation of gene body transcription at 24 hrs of BI-1347 treatment. C. Heatmap of Log 2 transformed fold change values of PRO-seq pausing indices of genes with changes in gene body transcription at the 24 hr time point. D. Heatmap showing log 2 transformed fold change of RNA-seq read counts for indicated gene sets after DMSO and BI-1347 (10 nM) treatment for 24 or 72 hrs. E. Bubble dot plot shows Gene Set Enrichment Analysis (GSEA) of PRO-seq gene hits that are upregulated at 24 hrs. Dot size indicates number of genes in each gene set, and dot color indicates p -adjusted value. F. Immunofluorescence analysis of myogenin (green; 20X) in Rh30 (left) and Rh4 (right) cells after 7 days of DMSO or BI-1347 treatment. G. Representative images of H&E stain of xenograft tumors. Arrows indicate myofibrils. H. Volcano plots reveal the number of changes in enhancer RNA (eRNA) transcripts after 24 hrs of BI-1347 treatment. Significantly upregulated eRNAs are highlighted in red; significantly downregulated eRNAs are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5) I. MA plots showing the changes of chromatin accessibility by ATAC-seq following 4 and 24 hrs of BI-1347 treatment. Significantly increased ATAC-seq peaks are highlighted in red; significantly decreased ATAC-seq peaks are highlighted in black (padj<0.05, fold change>1.5 or <-1.5). J. Pie chart showing the peak annotation of up-regulated ATAC-seq peaks to gene features at 24 hrs of BI-1347 treatment. K. Histogram of PRO-seq reads around up-regulated ATAC-seq peaks at indicated time points of BI-1347 treatment.

Techniques Used: Transformation Assay, RNA Sequencing, Immunofluorescence, Staining

A. IGV gene tracks showing a time course analysis of BI-1347 treatment by PRO-seq signal at the PAX3::FOXO1 locus. B. Heatmaps of CUT&RUN analysis of CDK8 and PAX3::FOXO1 binding in Rh30 and Rh4 cell lines. C. MA plots showing changes of PAX3::FOXO1 binding following BI-1347 treatment at 24 and 72 hrs. Significantly increased PAX3::FOXO1 peaks are highlighted in red; significantly decreased PAX3::FOXO1 peaks are highlighted in black (padj<0.05, fold change>1.5 or <-1.5). D, E. Heatmaps of CDK8 (D) and PAX3::FOXO1 (E) signal around PAX3::FOXO1-regulated enhancers before and after 24 hrs of BI-1347 treatment. F. Heatmaps of log 2 transformed fold change of RNA-seq read counts plotted by the shared gene between 24 hrs of PAX3::FOXO1 degradation and 72 hrs of CDK8 inhibition at the indicated time points. G. IGV gene tracks showing a time course analysis for PRO-seq signal at the VGLL2 locus after BI-1347 treatment.

Figure Legend Snippet: A. IGV gene tracks showing a time course analysis of BI-1347 treatment by PRO-seq signal at the PAX3::FOXO1 locus. B. Heatmaps of CUT&RUN analysis of CDK8 and PAX3::FOXO1 binding in Rh30 and Rh4 cell lines. C. MA plots showing changes of PAX3::FOXO1 binding following BI-1347 treatment at 24 and 72 hrs. Significantly increased PAX3::FOXO1 peaks are highlighted in red; significantly decreased PAX3::FOXO1 peaks are highlighted in black (padj<0.05, fold change>1.5 or <-1.5). D, E. Heatmaps of CDK8 (D) and PAX3::FOXO1 (E) signal around PAX3::FOXO1-regulated enhancers before and after 24 hrs of BI-1347 treatment. F. Heatmaps of log 2 transformed fold change of RNA-seq read counts plotted by the shared gene between 24 hrs of PAX3::FOXO1 degradation and 72 hrs of CDK8 inhibition at the indicated time points. G. IGV gene tracks showing a time course analysis for PRO-seq signal at the VGLL2 locus after BI-1347 treatment.

Techniques Used: Binding Assay, Transformation Assay, RNA Sequencing, Inhibition

A. Scatter plot showing the Z-scored average log2 fold change (LFC) of gene knockout effects in Rh30 cells treated with BI-1347 versus DMSO at day 14 (y-axis) and day 21 (x-axis) from genome-wide CRISPR-Cas9 screens. Each point represents an individual gene; dot size corresponds to statistical significance, and dot color indicates classification. B. Bubble dot plot of GSEA for gene hits scoring at day 21 in the CRISPR-Cas9 BI-1347 drug modifier screen ranked by C5 gene sets. C. Box plots showing construct-level Z-score averages for individual genes in the SAGA complex from the genome-wide CRISPR-Cas9 screen in Rh30 cells treated with DMSO (gray/black) or BI-1347 (blue/red) for 14 days (gray and blue) or 21 days (black and red). Genes are grouped by SAGA functional modules. D-F. Live cell proliferation assessed by Incucyte for BI-1347+/-sgTADA2B ( D ), BI-1347+/-sgTAF5L ( E ), and BI-1347+/-GSK699 ( F ). G. Quantitative real-time TaqMan qPCR analysis of RUNX1 , SEMA3D , and VGLL 2 expression at day 7 following treatment with vehicle control (DMSO), CDK8 inhibitors, or GSK699, and the indicated combination treatments. Expression levels were normalized to GAPDH gene expression and shown relative to DMSO control. Data represent means ± SEM (n=6).

Figure Legend Snippet: A. Scatter plot showing the Z-scored average log2 fold change (LFC) of gene knockout effects in Rh30 cells treated with BI-1347 versus DMSO at day 14 (y-axis) and day 21 (x-axis) from genome-wide CRISPR-Cas9 screens. Each point represents an individual gene; dot size corresponds to statistical significance, and dot color indicates classification. B. Bubble dot plot of GSEA for gene hits scoring at day 21 in the CRISPR-Cas9 BI-1347 drug modifier screen ranked by C5 gene sets. C. Box plots showing construct-level Z-score averages for individual genes in the SAGA complex from the genome-wide CRISPR-Cas9 screen in Rh30 cells treated with DMSO (gray/black) or BI-1347 (blue/red) for 14 days (gray and blue) or 21 days (black and red). Genes are grouped by SAGA functional modules. D-F. Live cell proliferation assessed by Incucyte for BI-1347+/-sgTADA2B ( D ), BI-1347+/-sgTAF5L ( E ), and BI-1347+/-GSK699 ( F ). G. Quantitative real-time TaqMan qPCR analysis of RUNX1 , SEMA3D , and VGLL 2 expression at day 7 following treatment with vehicle control (DMSO), CDK8 inhibitors, or GSK699, and the indicated combination treatments. Expression levels were normalized to GAPDH gene expression and shown relative to DMSO control. Data represent means ± SEM (n=6).

Techniques Used: Gene Knockout, Genome Wide, CRISPR, Construct, Functional Assay, Expressing, Control, Gene Expression

$A. Live cell proliferation assay by Incucyte for BI-1347 treatment combined with CRISPR knockout of SIX4 with two different guide RNAs. B. Western blot analysis of SIX4 protein level after treatment with three CDK8 inhibitors at indicated time points. Lamin B included as a loading control. C, D. Western blot analysis of SIX4 protein levels from cytoplasmic and nuclear fractions from Rh30 ( C ) and Rh4 ( D ) cells treated with DMSO or BI-1347 for 14 days. GAPDH and Lamin B served as nuclear and cytoplasmic loading controls, respectively. E, F. Volcano showing log 2 fold change of SIX4 ( E ) and TADA2B ( F ) genome binding sites determined by CUT&RUN analysis. Red highlights significantly up regulated sites and blue highlights significantly down regulated sites (log 2 FC 1.5, padj<0.05). G, H. Pie charts showing the annotation of upregulated CUT&RUN peaks for SIX4 ( G ) and TADA2B ( H ) sites after 24 hrs of BI-1347 treatment in Rh30 cells. I. Heatmaps of time course analysis of ATAC-seq signal, SIX4 signal, TADA2B signal, H3K27ac signal around upregulated ATAC-seq peaks at 24 hrs of BI-1347 treatment. J. IGV gene tracks showing the PRO-seq, SIX4, TADA2B, ATAC-seq, PAX3::FOXO1, CDK8, H3K4me3, and H3K27ac at the VGLL2 gene body and enhancers loci at indicated time points of BI-1347 treatment.$
Figure Legend Snippet: A. Live cell proliferation assay by Incucyte for BI-1347 treatment combined with CRISPR knockout of SIX4 with two different guide RNAs. B. Western blot analysis of SIX4 protein level after treatment with three CDK8 inhibitors at indicated time points. Lamin B included as a loading control. C, D. Western blot analysis of SIX4 protein levels from cytoplasmic and nuclear fractions from Rh30 ( C ) and Rh4 ( D ) cells treated with DMSO or BI-1347 for 14 days. GAPDH and Lamin B served as nuclear and cytoplasmic loading controls, respectively. E, F. Volcano showing log 2 fold change of SIX4 ( E ) and TADA2B ( F ) genome binding sites determined by CUT&RUN analysis. Red highlights significantly up regulated sites and blue highlights significantly down regulated sites (log 2 FC 1.5, padj<0.05). G, H. Pie charts showing the annotation of upregulated CUT&RUN peaks for SIX4 ( G ) and TADA2B ( H ) sites after 24 hrs of BI-1347 treatment in Rh30 cells. I. Heatmaps of time course analysis of ATAC-seq signal, SIX4 signal, TADA2B signal, H3K27ac signal around upregulated ATAC-seq peaks at 24 hrs of BI-1347 treatment. J. IGV gene tracks showing the PRO-seq, SIX4, TADA2B, ATAC-seq, PAX3::FOXO1, CDK8, H3K4me3, and H3K27ac at the VGLL2 gene body and enhancers loci at indicated time points of BI-1347 treatment.

Techniques Used: Proliferation Assay, CRISPR, Knock-Out, Western Blot, Control, Binding Assay

A. Box plots showing construct-level Z-score averages for individual genes in the Mediator complex from a genome-wide CRISPR-Cas9 screen in Rh30 cells treated with DMSO (gray/black) or BI-1347 (blue/red) for 14 days (gray and blue) or 21 days (black and red). Genes are grouped by Mediator functional modules. B. Live cell proliferation assessed by Incucyte for BI-1347+/-sgCDK8 (red) and BI-1347+/-sgCCNC (blue). C. MA plot showing changes of CDK8 binding site assessed by CUT&RUN after 24 hrs of BI-1347 treatment. Significantly increased CDK8 peaks are highlighted in red; significantly decreased CDK8 peaks are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5). D. Motif analysis of the regions with increased CDK8 DNA binding peaks from CUT&RUN analysis in Rh30 cells. E. Heatmaps showing chromatin occupancy of CDK8, CCNC, MED12, and MED13 at regions with upregulated SIX4 binding at 24 hrs of DMSO or BI-1347 treatment. F. IGV gene tracks showing the PRO-seq, CDK8, CCNC, MED12, and MED13 binding at the RUNX1 gene body and enhancer loci at indicated time points after BI-1347 treatment. G. Heatmaps of CDK8, CCNC, MED12, and MED13 CUT&RUN signal around PAX3::FOXO1-regulated enhancers before and after 24 hrs of BI-1347 treatment. H. IGV gene tracks showing the binding of CDK8, CCNC, MED12, and MED13 at a RUNX2 super enhancer cluster at indicated time points after BI-1347 treatment.

Figure Legend Snippet: A. Box plots showing construct-level Z-score averages for individual genes in the Mediator complex from a genome-wide CRISPR-Cas9 screen in Rh30 cells treated with DMSO (gray/black) or BI-1347 (blue/red) for 14 days (gray and blue) or 21 days (black and red). Genes are grouped by Mediator functional modules. B. Live cell proliferation assessed by Incucyte for BI-1347+/-sgCDK8 (red) and BI-1347+/-sgCCNC (blue). C. MA plot showing changes of CDK8 binding site assessed by CUT&RUN after 24 hrs of BI-1347 treatment. Significantly increased CDK8 peaks are highlighted in red; significantly decreased CDK8 peaks are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5). D. Motif analysis of the regions with increased CDK8 DNA binding peaks from CUT&RUN analysis in Rh30 cells. E. Heatmaps showing chromatin occupancy of CDK8, CCNC, MED12, and MED13 at regions with upregulated SIX4 binding at 24 hrs of DMSO or BI-1347 treatment. F. IGV gene tracks showing the PRO-seq, CDK8, CCNC, MED12, and MED13 binding at the RUNX1 gene body and enhancer loci at indicated time points after BI-1347 treatment. G. Heatmaps of CDK8, CCNC, MED12, and MED13 CUT&RUN signal around PAX3::FOXO1-regulated enhancers before and after 24 hrs of BI-1347 treatment. H. IGV gene tracks showing the binding of CDK8, CCNC, MED12, and MED13 at a RUNX2 super enhancer cluster at indicated time points after BI-1347 treatment.

Techniques Used: Construct, Genome Wide, CRISPR, Functional Assay, Binding Assay

Image Search Results

The overexpression of CDK8 exacerbates knee cartilage degeneration and pain in mice, as well as osteoarthritis in cells. (A) H&E and S-O staining of the knee joints in each group of mice 8 weeks after DMM surgery, (B) followed by assessment of OA severity in the mice using the OARSI scoring system. n = 10 per group. (C) Aggrecan, COL2A1, and MMP-3 expression were detected by immunohistochemical staining in the cartilage samples, (D-F) and quantitative analysis of the proportion of Aggrecan, COL2A1, and MMP-3 positive cells in each segment. n = 6 per group. (G-H) The Pressure Application Measurement (PAM) test and Von Frey filament test were used to assess the pain threshold in mice. (I) C28/I2 cells were transfected with pCMV-control or pCMV-CDK8 and then exposed to IL-1β for 48 h. Western blot analysis was performed to detect aggrecan, COL2A1, SOX9, MMP-3, and MMP-13. (J) C28/I2 and mouse chondrocytes were transfected with pCMV-CDK8 and treated with IL-1β for 7 days. Safranin O and Toluidine Blue staining were used to assess cartilage formation, (K) and the culture supernatants from the last two days were collected to analyze cartilage degradation using the DMMB method to measure GAG content. Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Journal: Journal of Advanced Research

Article Title: CDK8 mediated inflammatory microenvironment aggravates osteoarthritis progression

doi: 10.1016/j.jare.2025.01.017

Figure Lengend Snippet: The overexpression of CDK8 exacerbates knee cartilage degeneration and pain in mice, as well as osteoarthritis in cells. (A) H&E and S-O staining of the knee joints in each group of mice 8 weeks after DMM surgery, (B) followed by assessment of OA severity in the mice using the OARSI scoring system. n = 10 per group. (C) Aggrecan, COL2A1, and MMP-3 expression were detected by immunohistochemical staining in the cartilage samples, (D-F) and quantitative analysis of the proportion of Aggrecan, COL2A1, and MMP-3 positive cells in each segment. n = 6 per group. (G-H) The Pressure Application Measurement (PAM) test and Von Frey filament test were used to assess the pain threshold in mice. (I) C28/I2 cells were transfected with pCMV-control or pCMV-CDK8 and then exposed to IL-1β for 48 h. Western blot analysis was performed to detect aggrecan, COL2A1, SOX9, MMP-3, and MMP-13. (J) C28/I2 and mouse chondrocytes were transfected with pCMV-CDK8 and treated with IL-1β for 7 days. Safranin O and Toluidine Blue staining were used to assess cartilage formation, (K) and the culture supernatants from the last two days were collected to analyze cartilage degradation using the DMMB method to measure GAG content. Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used are listed as follows: Aggrecan (HuaBio, Cat. ET1704-57), COL2A1 (Abcam, Cat. ab34712), SOX9 (HuaBio, Cat. et1611-56), MMP3 (Abcam, Cat. ab52915), MMP13 (Abcam, Cat. ab39012), CDK8 (Proteintech, Cat. 22067–1-AP), p65 (CST, Cat. 8242), p-p65 (Affinity, Cat. AF2006), IκBα (CST, Cat. 4814), p-IκBα (CST, Cat. 2859), Rpb1 CTD (CST, Cat. 2629), p-Rpb1 CTD(ser 2)(CST, Cat. 13499), p-Rpb1 CTD(ser 5)(CST, Cat. 13523), Cleaved Caspase-1(CST, Cat. 89332), Cleaved-IL-1β (CST, Cat. 63124), Caspase1 (HuaBio, Cat. ET1608-69), NLRP3 (Abcam, Cat. ab214185), ASC (Proteintech, Cat. 10500–1-AP), FLAG (HuaBio, Cat. 0912–1), β-Actin (Fudebio, Cat. FD0060), Histone H3 (Proteintech, Cat. 68345–1-Ig), IgG (Proteintech, Cat. 30000–0-AP).

Techniques: Over Expression, Staining, Expressing, Immunohistochemical staining, Transfection, Control, Western Blot

CDK8 knockdown alleviates knee cartilage degeneration and pain in mice and reduces osteoarthritis in cells. (A-B) C28/I2 cells and mouse chondrocytes were transfected with si-CDK8 or si-NC and then exposed to IL-1β for 48 h. (A) qRT-PCR and (B) Western blot analyses were used to detect the expression levels of aggrecan, COL2A1, SOX9, MMP-3, and MMP-13. (C) IF analysis was used to detect the expression of aggrecan and COL2A1 in C28/I2 cells transfected with si-CDK8 or si-NC and exposed to IL-1β for 48 h. (D, E) Safranin O and Toluidine Blue staining were used to assess cartilage formation, analyzed in (D) C28/I2 cells or (E) mouse chondrocytes, both transfected with si-CDK8 and treated with IL-1β for 7 days. (F) Hematoxylin and Eosin (H&E) staining and Safranin O/Fast Green staining of mouse knee joints 8 weeks after DMM surgery, and (G) the severity of osteoarthritis in mice was subsequently assessed using the OARSI scoring system. Each group, n = 6. (H) Pain threshold in mice was assessed using the pressure application measurement (PAM) test. Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Journal: Journal of Advanced Research

Article Title: CDK8 mediated inflammatory microenvironment aggravates osteoarthritis progression

doi: 10.1016/j.jare.2025.01.017

Figure Lengend Snippet: CDK8 knockdown alleviates knee cartilage degeneration and pain in mice and reduces osteoarthritis in cells. (A-B) C28/I2 cells and mouse chondrocytes were transfected with si-CDK8 or si-NC and then exposed to IL-1β for 48 h. (A) qRT-PCR and (B) Western blot analyses were used to detect the expression levels of aggrecan, COL2A1, SOX9, MMP-3, and MMP-13. (C) IF analysis was used to detect the expression of aggrecan and COL2A1 in C28/I2 cells transfected with si-CDK8 or si-NC and exposed to IL-1β for 48 h. (D, E) Safranin O and Toluidine Blue staining were used to assess cartilage formation, analyzed in (D) C28/I2 cells or (E) mouse chondrocytes, both transfected with si-CDK8 and treated with IL-1β for 7 days. (F) Hematoxylin and Eosin (H&E) staining and Safranin O/Fast Green staining of mouse knee joints 8 weeks after DMM surgery, and (G) the severity of osteoarthritis in mice was subsequently assessed using the OARSI scoring system. Each group, n = 6. (H) Pain threshold in mice was assessed using the pressure application measurement (PAM) test. Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Staining, Control

CDK8 is associated with senescence in chondrocytes and regulate their secretion of SASP. (A) Volcano plot of differentially expressed genes from RNA sequencing data. (B) KEGG enrichment analysis of differentially expressed genes. (C-D) GSEA of differentially expressed genes. (E) β-galactosidase staining images of mouse chondrocytes with CDK8 knockdown, co-treated with t-BHP. (F) Representative knee X-rays of patients at different stages of osteoarthritis. According to the Kellgren-Lawrence (KL) grading of the tibiofemoral joint, patients were classified into four stages. Stage I is classified as mild osteoarthritis, n = 23; middle osteoarthritis included stages II and III, n = 29; and stage IV was defined as severe osteoarthritis, n = 35. (G-I) ELISA detection of SASP levels in (G) synovial fluid of patients with mild, moderate, and severe OA, (H) serum of mice, and (I) C28/I2 cells and mouse chondrocytes. Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated.

Journal: Journal of Advanced Research

Article Title: CDK8 mediated inflammatory microenvironment aggravates osteoarthritis progression

doi: 10.1016/j.jare.2025.01.017

Figure Lengend Snippet: CDK8 is associated with senescence in chondrocytes and regulate their secretion of SASP. (A) Volcano plot of differentially expressed genes from RNA sequencing data. (B) KEGG enrichment analysis of differentially expressed genes. (C-D) GSEA of differentially expressed genes. (E) β-galactosidase staining images of mouse chondrocytes with CDK8 knockdown, co-treated with t-BHP. (F) Representative knee X-rays of patients at different stages of osteoarthritis. According to the Kellgren-Lawrence (KL) grading of the tibiofemoral joint, patients were classified into four stages. Stage I is classified as mild osteoarthritis, n = 23; middle osteoarthritis included stages II and III, n = 29; and stage IV was defined as severe osteoarthritis, n = 35. (G-I) ELISA detection of SASP levels in (G) synovial fluid of patients with mild, moderate, and severe OA, (H) serum of mice, and (I) C28/I2 cells and mouse chondrocytes. Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated.

Techniques: RNA Sequencing, Staining, Knockdown, Enzyme-linked Immunosorbent Assay, Control

CDK8 activates the NF-κB pathway and regulates the transcription of SASP. (A) Western blot assay was used to detect the expression levels of p-IκBα, IκBα, p-p65, and p65. (B) Immunoprecipitation was performed to assess the co-precipitation of CDK8 and p65 in C28/I2 cells treated with or without CDK8-IN-6. (C) Immunofluorescence co-localization was conducted to examine the co-localization of p65 and CDK8 in C28/I2 cells treated with IL-1β for 24 h. (D) C28/I2 cells were transfected with si-CDK8 or pCMV3-CDK8, followed by IL-1β treatment for 24 h, and then subjected to western blot analysis of nuclear and cytoplasmic proteins. (E-F) Dual-luciferase analysis demonstrated NF-κB's regulatory role in SASP transcriptional activation (E) and confirmed the direct binding sites of p65 in the promoters of the IL-6, IL-8, and MMP-13 genes (F), the statistical results indicating the significance of the differences between the truncated promoter groups and the full-length group. (G) Nucleic acid electrophoresis and (H) ChIP-qPCR further investigated the influence of CDK8 on the binding of p65 to SASP promoter region sites in C28/I2 cells. (I) Western blot assay was used to detect the expression levels of Rpb1 CTD, p-Rpb1 CTD (Ser2), and p-Rpb1 CTD (Ser5). Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated.

Journal: Journal of Advanced Research

Article Title: CDK8 mediated inflammatory microenvironment aggravates osteoarthritis progression

doi: 10.1016/j.jare.2025.01.017

Figure Lengend Snippet: CDK8 activates the NF-κB pathway and regulates the transcription of SASP. (A) Western blot assay was used to detect the expression levels of p-IκBα, IκBα, p-p65, and p65. (B) Immunoprecipitation was performed to assess the co-precipitation of CDK8 and p65 in C28/I2 cells treated with or without CDK8-IN-6. (C) Immunofluorescence co-localization was conducted to examine the co-localization of p65 and CDK8 in C28/I2 cells treated with IL-1β for 24 h. (D) C28/I2 cells were transfected with si-CDK8 or pCMV3-CDK8, followed by IL-1β treatment for 24 h, and then subjected to western blot analysis of nuclear and cytoplasmic proteins. (E-F) Dual-luciferase analysis demonstrated NF-κB's regulatory role in SASP transcriptional activation (E) and confirmed the direct binding sites of p65 in the promoters of the IL-6, IL-8, and MMP-13 genes (F), the statistical results indicating the significance of the differences between the truncated promoter groups and the full-length group. (G) Nucleic acid electrophoresis and (H) ChIP-qPCR further investigated the influence of CDK8 on the binding of p65 to SASP promoter region sites in C28/I2 cells. (I) Western blot assay was used to detect the expression levels of Rpb1 CTD, p-Rpb1 CTD (Ser2), and p-Rpb1 CTD (Ser5). Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated.

Techniques: Western Blot, Expressing, Immunoprecipitation, Immunofluorescence, Transfection, Luciferase, Activation Assay, Binding Assay, Nucleic Acid Electrophoresis, ChIP-qPCR, Control

CDK8 and NF-κB are cooperatively recruited to SASP promoters, leading to elongation phosphorylation of the Rpb1 CTD. In C28/I2 cells transfected with si-NC or si-CDK8, with or without IL-1β treatment for one hour, (A–E) ChIP analysis shows the effects of CDK8 knockdown and IL-1β treatment on the binding of p65 (A), CDK8 (B), Rpb1 CTD (C), Rpb1 CTD phosphorylated at Ser5 (D), and Rpb1 CTD phosphorylated at Ser2 (E) to three SASP genes and a housekeeping gene. The gene diagrams are displayed at the top.

Journal: Journal of Advanced Research

Article Title: CDK8 mediated inflammatory microenvironment aggravates osteoarthritis progression

doi: 10.1016/j.jare.2025.01.017

Figure Lengend Snippet: CDK8 and NF-κB are cooperatively recruited to SASP promoters, leading to elongation phosphorylation of the Rpb1 CTD. In C28/I2 cells transfected with si-NC or si-CDK8, with or without IL-1β treatment for one hour, (A–E) ChIP analysis shows the effects of CDK8 knockdown and IL-1β treatment on the binding of p65 (A), CDK8 (B), Rpb1 CTD (C), Rpb1 CTD phosphorylated at Ser5 (D), and Rpb1 CTD phosphorylated at Ser2 (E) to three SASP genes and a housekeeping gene. The gene diagrams are displayed at the top.

Techniques: Phospho-proteomics, Transfection, Knockdown, Binding Assay

CDK8 promotes the inflammatory microenvironment and osteoclast differentiation of macrophages by regulating the SASP in chondrocytes. (A) Schematic diagram of the migration assay for RAW 264.7 cells. (B-C) Cell migration ability was evaluated using the migration assay (top) and quantification of migrated cells (bottom). (D-E) Western blotting was used to assess the impact of CDK8 on inflammasome activation in murine synovial macrophages. (F-G) The degree of synovitis was evaluated using H&E-stained sections of mouse knee joints. (H and J) TRAP staining was used to detect the extent of osteoclast differentiation. (I and K) The number and average area of TRAP-positive multinucleated osteoclasts were quantified. The number of TRAP-positive osteoclasts with 3–5, 5–10, or more than 10 nuclei was also determined. (L-M) Bone destruction was assessed by microCT of mouse knee joints, with arrows in the figure indicating the medial side of the knee joints. Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated.

Journal: Journal of Advanced Research

Article Title: CDK8 mediated inflammatory microenvironment aggravates osteoarthritis progression

doi: 10.1016/j.jare.2025.01.017

Figure Lengend Snippet: CDK8 promotes the inflammatory microenvironment and osteoclast differentiation of macrophages by regulating the SASP in chondrocytes. (A) Schematic diagram of the migration assay for RAW 264.7 cells. (B-C) Cell migration ability was evaluated using the migration assay (top) and quantification of migrated cells (bottom). (D-E) Western blotting was used to assess the impact of CDK8 on inflammasome activation in murine synovial macrophages. (F-G) The degree of synovitis was evaluated using H&E-stained sections of mouse knee joints. (H and J) TRAP staining was used to detect the extent of osteoclast differentiation. (I and K) The number and average area of TRAP-positive multinucleated osteoclasts were quantified. The number of TRAP-positive osteoclasts with 3–5, 5–10, or more than 10 nuclei was also determined. (L-M) Bone destruction was assessed by microCT of mouse knee joints, with arrows in the figure indicating the medial side of the knee joints. Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated.

Techniques: Migration, Western Blot, Activation Assay, Staining, Control

CDK8 inhibitors alleviated the progression of OA. (A) Molecular structure of CDK8-IN-6. (B) Use the Cell Counting Kit-8 (CCK-8) assay to evaluate the cytotoxic effects of CDK8-IN-6 on C28/I2 cells and mouse chondrocytes at 24 and 48 h. (C-D) C28/I2 cells and mouse chondrocytes were co-treated with IL-1β and CDK8-IN-6 for 48 h. (C) qRT-PCR and (D) Western blot analyses were performed to detect the expression of aggrecan, COL2A1, SOX9, MMP-3, and MMP-13. (E) Immunofluorescence analysis was used to determine the expression of aggrecan and COL2A1 in C28/I2 cells co-treated with IL-1β and CDK8-IN-6 for 48 h. (F, H) Safranin O and Toluidine Blue staining were used to assess cartilage formation in (F) C28/I2 cells and (H) mouse chondrocytes, both treated with IL-1β and CDK8-IN-6 for 7 days. (G, I) The collected supernatants were used to detect GAG content. Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Journal: Journal of Advanced Research

Article Title: CDK8 mediated inflammatory microenvironment aggravates osteoarthritis progression

doi: 10.1016/j.jare.2025.01.017

Figure Lengend Snippet: CDK8 inhibitors alleviated the progression of OA. (A) Molecular structure of CDK8-IN-6. (B) Use the Cell Counting Kit-8 (CCK-8) assay to evaluate the cytotoxic effects of CDK8-IN-6 on C28/I2 cells and mouse chondrocytes at 24 and 48 h. (C-D) C28/I2 cells and mouse chondrocytes were co-treated with IL-1β and CDK8-IN-6 for 48 h. (C) qRT-PCR and (D) Western blot analyses were performed to detect the expression of aggrecan, COL2A1, SOX9, MMP-3, and MMP-13. (E) Immunofluorescence analysis was used to determine the expression of aggrecan and COL2A1 in C28/I2 cells co-treated with IL-1β and CDK8-IN-6 for 48 h. (F, H) Safranin O and Toluidine Blue staining were used to assess cartilage formation in (F) C28/I2 cells and (H) mouse chondrocytes, both treated with IL-1β and CDK8-IN-6 for 7 days. (G, I) The collected supernatants were used to detect GAG content. Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Techniques: Cell Counting, CCK-8 Assay, Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence, Staining, Control

CDK8-IN-6 selectively inhibits CDK8 to alleviate OA. (A) Docking-predicted binding mode of CDK8 protein with CDK8-IN-6. The overall structure of CDK8 in complex with CDK8-IN-6 in cartoon view. CDK8 and CDK8-IN-6 are colored green and sky blue, respectively (top). Detailed interaction network between CDK8 and CDK8-IN-6, showing all potential binding sites (bottom). (B) Proteins extracted from C28/I2 cells and mouse chondrocytes were used to evaluate the binding of CDK8-IN-6 to CDK8 protein at different concentrations of CDK8-IN-6 and Pronase in DARTS experiments. The binding of CDK8-IN-6 to CDK8 protein was detected by Western blotting. (C) C28/I2 cells were transfected with the indicated CDK8 mutant plasmids for 24 h and subjected to DARTS experiments. Protein expression of the mutated sites was then detected by Western blotting (bottom) and quantified (top). (D) At 8 weeks post-DMM surgery, knee joints from each group of mice were stained with H&E and S-O. (E) Subsequently, the severity of osteoarthritis in mice was assessed using the OARSI scoring system. Each group had n = 10. (F) Expression of Aggrecan, COL2A1, and MMP-3 in cartilage samples was detected by immunohistochemical staining, (G-I) and the proportion of Aggrecan, COL2A1, and MMP-3 positive cells in each section was quantified. Each group had n = 6. (J) Pressure Application Measurement (PAM) test was used to assess the pain threshold in mice. Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Journal: Journal of Advanced Research

Article Title: CDK8 mediated inflammatory microenvironment aggravates osteoarthritis progression

doi: 10.1016/j.jare.2025.01.017

Figure Lengend Snippet: CDK8-IN-6 selectively inhibits CDK8 to alleviate OA. (A) Docking-predicted binding mode of CDK8 protein with CDK8-IN-6. The overall structure of CDK8 in complex with CDK8-IN-6 in cartoon view. CDK8 and CDK8-IN-6 are colored green and sky blue, respectively (top). Detailed interaction network between CDK8 and CDK8-IN-6, showing all potential binding sites (bottom). (B) Proteins extracted from C28/I2 cells and mouse chondrocytes were used to evaluate the binding of CDK8-IN-6 to CDK8 protein at different concentrations of CDK8-IN-6 and Pronase in DARTS experiments. The binding of CDK8-IN-6 to CDK8 protein was detected by Western blotting. (C) C28/I2 cells were transfected with the indicated CDK8 mutant plasmids for 24 h and subjected to DARTS experiments. Protein expression of the mutated sites was then detected by Western blotting (bottom) and quantified (top). (D) At 8 weeks post-DMM surgery, knee joints from each group of mice were stained with H&E and S-O. (E) Subsequently, the severity of osteoarthritis in mice was assessed using the OARSI scoring system. Each group had n = 10. (F) Expression of Aggrecan, COL2A1, and MMP-3 in cartilage samples was detected by immunohistochemical staining, (G-I) and the proportion of Aggrecan, COL2A1, and MMP-3 positive cells in each section was quantified. Each group had n = 6. (J) Pressure Application Measurement (PAM) test was used to assess the pain threshold in mice. Data are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no significance; comparisons with the control group or as indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Techniques: Binding Assay, Western Blot, Transfection, Mutagenesis, Expressing, Staining, Immunohistochemical staining, Control

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Volcano plot of ssGSEA on genome-wide differential effect size of CORUM complexes comparing aRMS to other non-RMS tumor cell lines. Red indicates Mediator complex. B. Distribution of CDK8 gene effect score across different cancer cell lines from the Broad Institute’s CRISPR Dependency Map (24Q2). C. Dot plot of kinase dependencies in the Broad Institute’s CRISPR Dependency Map comparing fusion-positive RMS to all other cancer cell lines. CDK8 is highlighted in red. D. Violin plots showing distribution of CCNC , MED13 , and MED12 gene effect score from the Broad Institute’s CRISPR Dependency Map (24Q2) comparing the fusion-positive aRMS and fusion-negative eRMS with all other indicated cancer cell lines. aRMS is highlighted in red and eRMS is highlighted in blue. E. shRNA-mediated suppression of CDK8 by two different shRNAs impairs Rh30 and Rh28 aRMS cell growth in vitro . Cell numbers were determined by trypan blue live cell counting. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). F. Line graph showing mean subcutaneous tumor volume (mm3) formed by Rh28 cells after treatment with inducible knock down of CDK8 using shRNA. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). G. CRISPR-mediated knockout of CDK8 by two different gRNAs impairs Rh30 and Rh4 aRMS cell growth in vitro . Relative growth was assessed by CellTiter-Glo after CRISPR knockout. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04).

Article Snippet: Primary antibodies used in this study includes: CDK8 (ProteinTech, #22067; Cell Signaling Technology, #4106S; Santa Cruz Biotechnology, #SC-13155; abcam, #ab229192), STAT1 (Cell Signaling Technology, #9176S), pSTAT1S727(Cell Signaling Technology, #8826S), SIX4 (Santa Cruz Biotechnology, #SC-390779), GAPDH (Santa Cruz Biotechnology, #SC-365062), LaminB (Abcam, #ab16048; Santa Cruz Biotechnology, #SC-374015), ACTB (Sigma Aldrich, #A2066), full PARP (Cell Signaling Technology, #9542S), cleaved PARP (Cell Signaling Technology, #5625S), PAX3::FOXO1 (CancerTools, #160866), MYC (XXX), streptavidin (XXX), TAF5L (ProteinTech, #19274), TADA2B (ProteinTech, #17367), KAT2A (ProteinTech, #66575), KAT2B (Cell Signaling Technology, #C14G9), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028).

Techniques: Genome Wide, CRISPR, shRNA, In Vitro, Cell Counting, Knockdown, Knock-Out

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Correlation between CDK8 gene dependency and sensitivity to the CDK8 inhibitor BI-1347 in 669 cancer cell lines (y-axis showing CDK8 dependency score; x-axis showing PRISM LFC value of BI-1347 treatment). Linear regression lines and Pearson correlation coefficients (R) are shown for all cell lines (black), aRMS (red), and eRMS (blue). B. Western blot analysis showing CDK8 inhibition by small molecules as assessed by STAT1 phosphorylation at serine 727. C, D. Dose response curves of inactive BI-1347 analog (BI-1374) (C) and three pharmacologic CDK8 inhibitors (D), BI-1347, SEL-120-34A, and JH-XII-178, at day 7 of treatment in Rh30, Rh4 and RHJT cell lines. E, F. Live cell proliferation assessed by Incucyte for Rh30 (E) and Rh4 (F) cells after treatment with vehicle DMSO (black), the CDK8 inhibitor BI-1347 (red) and its inactive analog BI-1374 (gray). Data were normalized to DMSO. Data represent means ± SEM (n=6, *: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04). G. Line graph reveals mean subcutaneous tumor volume (mm ) formed by Rh30 cells after treatment with the CDK8 inhibitor SEL-120-34A. Data are presented as mean ± SEM (*: p <=5.0e-02, **: p <=1.0e-02, ***: p <= 1.0e-03, ****: p <=1.0e-04).

Techniques: Western Blot, Inhibition, Phospho-proteomics

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Volcano plots showing the number of gene body changes after 24 hrs of CDK8 inhibitor BI-1347 treatment. Significantly up-regulated genes are highlighted in red; significantly downregulated genes are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5). B. Metagene plots of PRO-seq reads of genes with significant upregulation or downregulation of gene body transcription at 24 hrs of BI-1347 treatment. C. Heatmap of Log 2 transformed fold change values of PRO-seq pausing indices of genes with changes in gene body transcription at the 24 hr time point. D. Heatmap showing log 2 transformed fold change of RNA-seq read counts for indicated gene sets after DMSO and BI-1347 (10 nM) treatment for 24 or 72 hrs. E. Bubble dot plot shows Gene Set Enrichment Analysis (GSEA) of PRO-seq gene hits that are upregulated at 24 hrs. Dot size indicates number of genes in each gene set, and dot color indicates p -adjusted value. F. Immunofluorescence analysis of myogenin (green; 20X) in Rh30 (left) and Rh4 (right) cells after 7 days of DMSO or BI-1347 treatment. G. Representative images of H&E stain of xenograft tumors. Arrows indicate myofibrils. H. Volcano plots reveal the number of changes in enhancer RNA (eRNA) transcripts after 24 hrs of BI-1347 treatment. Significantly upregulated eRNAs are highlighted in red; significantly downregulated eRNAs are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5) I. MA plots showing the changes of chromatin accessibility by ATAC-seq following 4 and 24 hrs of BI-1347 treatment. Significantly increased ATAC-seq peaks are highlighted in red; significantly decreased ATAC-seq peaks are highlighted in black (padj<0.05, fold change>1.5 or <-1.5). J. Pie chart showing the peak annotation of up-regulated ATAC-seq peaks to gene features at 24 hrs of BI-1347 treatment. K. Histogram of PRO-seq reads around up-regulated ATAC-seq peaks at indicated time points of BI-1347 treatment.

Techniques: Transformation Assay, RNA Sequencing, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. IGV gene tracks showing a time course analysis of BI-1347 treatment by PRO-seq signal at the PAX3::FOXO1 locus. B. Heatmaps of CUT&RUN analysis of CDK8 and PAX3::FOXO1 binding in Rh30 and Rh4 cell lines. C. MA plots showing changes of PAX3::FOXO1 binding following BI-1347 treatment at 24 and 72 hrs. Significantly increased PAX3::FOXO1 peaks are highlighted in red; significantly decreased PAX3::FOXO1 peaks are highlighted in black (padj<0.05, fold change>1.5 or <-1.5). D, E. Heatmaps of CDK8 (D) and PAX3::FOXO1 (E) signal around PAX3::FOXO1-regulated enhancers before and after 24 hrs of BI-1347 treatment. F. Heatmaps of log 2 transformed fold change of RNA-seq read counts plotted by the shared gene between 24 hrs of PAX3::FOXO1 degradation and 72 hrs of CDK8 inhibition at the indicated time points. G. IGV gene tracks showing a time course analysis for PRO-seq signal at the VGLL2 locus after BI-1347 treatment.

Techniques: Binding Assay, Transformation Assay, RNA Sequencing, Inhibition

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Scatter plot showing the Z-scored average log2 fold change (LFC) of gene knockout effects in Rh30 cells treated with BI-1347 versus DMSO at day 14 (y-axis) and day 21 (x-axis) from genome-wide CRISPR-Cas9 screens. Each point represents an individual gene; dot size corresponds to statistical significance, and dot color indicates classification. B. Bubble dot plot of GSEA for gene hits scoring at day 21 in the CRISPR-Cas9 BI-1347 drug modifier screen ranked by C5 gene sets. C. Box plots showing construct-level Z-score averages for individual genes in the SAGA complex from the genome-wide CRISPR-Cas9 screen in Rh30 cells treated with DMSO (gray/black) or BI-1347 (blue/red) for 14 days (gray and blue) or 21 days (black and red). Genes are grouped by SAGA functional modules. D-F. Live cell proliferation assessed by Incucyte for BI-1347+/-sgTADA2B ( D ), BI-1347+/-sgTAF5L ( E ), and BI-1347+/-GSK699 ( F ). G. Quantitative real-time TaqMan qPCR analysis of RUNX1 , SEMA3D , and VGLL 2 expression at day 7 following treatment with vehicle control (DMSO), CDK8 inhibitors, or GSK699, and the indicated combination treatments. Expression levels were normalized to GAPDH gene expression and shown relative to DMSO control. Data represent means ± SEM (n=6).

Techniques: Gene Knockout, Genome Wide, CRISPR, Construct, Functional Assay, Expressing, Control, Gene Expression

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Live cell proliferation assay by Incucyte for BI-1347 treatment combined with CRISPR knockout of SIX4 with two different guide RNAs. B. Western blot analysis of SIX4 protein level after treatment with three CDK8 inhibitors at indicated time points. Lamin B included as a loading control. C, D. Western blot analysis of SIX4 protein levels from cytoplasmic and nuclear fractions from Rh30 ( C ) and Rh4 ( D ) cells treated with DMSO or BI-1347 for 14 days. GAPDH and Lamin B served as nuclear and cytoplasmic loading controls, respectively. E, F. Volcano showing log 2 fold change of SIX4 ( E ) and TADA2B ( F ) genome binding sites determined by CUT&RUN analysis. Red highlights significantly up regulated sites and blue highlights significantly down regulated sites (log 2 FC 1.5, padj<0.05). G, H. Pie charts showing the annotation of upregulated CUT&RUN peaks for SIX4 ( G ) and TADA2B ( H ) sites after 24 hrs of BI-1347 treatment in Rh30 cells. I. Heatmaps of time course analysis of ATAC-seq signal, SIX4 signal, TADA2B signal, H3K27ac signal around upregulated ATAC-seq peaks at 24 hrs of BI-1347 treatment. J. IGV gene tracks showing the PRO-seq, SIX4, TADA2B, ATAC-seq, PAX3::FOXO1, CDK8, H3K4me3, and H3K27ac at the VGLL2 gene body and enhancers loci at indicated time points of BI-1347 treatment.

Techniques: Proliferation Assay, CRISPR, Knock-Out, Western Blot, Control, Binding Assay

Journal: bioRxiv

Article Title: CDK8 Inhibition Releases the Muscle Differentiation Block in Fusion-driven Alveolar Rhabdomyosarcoma

doi: 10.1101/2025.07.14.663986

Figure Lengend Snippet: A. Box plots showing construct-level Z-score averages for individual genes in the Mediator complex from a genome-wide CRISPR-Cas9 screen in Rh30 cells treated with DMSO (gray/black) or BI-1347 (blue/red) for 14 days (gray and blue) or 21 days (black and red). Genes are grouped by Mediator functional modules. B. Live cell proliferation assessed by Incucyte for BI-1347+/-sgCDK8 (red) and BI-1347+/-sgCCNC (blue). C. MA plot showing changes of CDK8 binding site assessed by CUT&RUN after 24 hrs of BI-1347 treatment. Significantly increased CDK8 peaks are highlighted in red; significantly decreased CDK8 peaks are highlighted in blue (padj<0.05, fold change>1.5 or <-1.5). D. Motif analysis of the regions with increased CDK8 DNA binding peaks from CUT&RUN analysis in Rh30 cells. E. Heatmaps showing chromatin occupancy of CDK8, CCNC, MED12, and MED13 at regions with upregulated SIX4 binding at 24 hrs of DMSO or BI-1347 treatment. F. IGV gene tracks showing the PRO-seq, CDK8, CCNC, MED12, and MED13 binding at the RUNX1 gene body and enhancer loci at indicated time points after BI-1347 treatment. G. Heatmaps of CDK8, CCNC, MED12, and MED13 CUT&RUN signal around PAX3::FOXO1-regulated enhancers before and after 24 hrs of BI-1347 treatment. H. IGV gene tracks showing the binding of CDK8, CCNC, MED12, and MED13 at a RUNX2 super enhancer cluster at indicated time points after BI-1347 treatment.

Techniques: Construct, Genome Wide, CRISPR, Functional Assay, Binding Assay

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