Journal: Cancer discovery

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

doi: 10.1158/2159-8290.CD-25-1171

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 plot of C5 gene sets enriched in GSEA at day 21 in the CRISPR-Cas9 BI-1347 drug modifier screen. C. Box and whisker 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. Boxes indicate the median and interquartile range (IQR; 25 th -75 th percentile); whiskers extend to 1.5x the IQR. Values beyond the end of the whiskers are plotted individually. D. Quantitative real-time TaqMan qPCR analysis of RUNX1 , SEMA3D , VGLL 2, and MYL1 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=3; Statistics analysis is showed in Table. S1G ). E-G. Live cell proliferation assessed by Incucyte for BI-1347+/−sgTADA2B ( E ), BI-1347+/−sgTAF5L ( F ), and BI-1347+/−GSK699 ( G ) in Rh30 cells. Data are presented as mean ± SEM (n=3; AUC *** p <=1.0e-03). H. Relative growth was assessed by CellTiter-Glo for Rh28 cells with the treatment of BI-1347+/−sgTADA2B and SEL-120–34A+/−sgTADA2B. Data are presented as mean ± SEM (n=6; AUC ** p <=1.0e-02). I. Line graph reveals mean subcutaneous tumor volume (mm 3 ) formed by Rh28_sgChr2.2 or Rh28_sgTADA2B cells after treatment with the CDK8 inhibitor SEL-120–34A. Data are presented as mean ± SEM (n_sgChr2.2-Vehicle=12, n_sgChr2.2-SEL-120–34A=11, n_sgTADA2B-Vehible=12, n_sgTADA2B-SEL-120–34A-12; AUC *: p <=5.0e-02).

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), total PARP (Cell Signaling Technology, #9542S), cleaved PARP (Cell Signaling Technology, #5625S), cleaved caspase 3 (Cell Signaling Technology, #9664S), FOXO1 (Cell Signaling Technology, #2380S), PAX3::FOXO1 (CancerTools, #160866), MYC (EMD Milipore, #05–724), Streptavidin-HRP (Cell Signaling Technology, #3999), TAF5L (ProteinTech, #19274), TADA2B (ProteinTech, #17367), KAT2A (ProteinTech, #66575), KAT2B (Cell Signaling Technology, #C14G9), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), MED1 (Bethyl Laboratories, #A300–793A), MEF2C (Abcam, #ab221493), RUNX1 (ProteinTech, #25315), MYL1 (ProteinTech, #15814), MF20 (Fisher Scientific, #MAB4470), FLAG (Sigma, #F3165).

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

Journal: Cancer discovery

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

doi: 10.1158/2159-8290.CD-25-1171

Figure Lengend Snippet: A. Box and whisker 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. Boxes indicate the median and interquartile range (IQR; 25 th -75 th percentile); whiskers extend to 1.5x the IQR. Values beyond the end of the whiskers are plotted individually. B. Live cell proliferation assessed by Incucyte for BI-1347+/−sgCCNC (blue) or sgCDK8 (red). Data represent means ± SEM (n=3; AUC **: p <=1.0e-02, ***: p <= 1.0e-03) 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, MED13, and MED1 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 MED1 binding at the RUNX1 gene body and enhancer loci at indicated time points after BI-1347 treatment.

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), total PARP (Cell Signaling Technology, #9542S), cleaved PARP (Cell Signaling Technology, #5625S), cleaved caspase 3 (Cell Signaling Technology, #9664S), FOXO1 (Cell Signaling Technology, #2380S), PAX3::FOXO1 (CancerTools, #160866), MYC (EMD Milipore, #05–724), Streptavidin-HRP (Cell Signaling Technology, #3999), TAF5L (ProteinTech, #19274), TADA2B (ProteinTech, #17367), KAT2A (ProteinTech, #66575), KAT2B (Cell Signaling Technology, #C14G9), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), MED1 (Bethyl Laboratories, #A300–793A), MEF2C (Abcam, #ab221493), RUNX1 (ProteinTech, #25315), MYL1 (ProteinTech, #15814), MF20 (Fisher Scientific, #MAB4470), FLAG (Sigma, #F3165).

Techniques: Activity Assay, Whisker Assay, Construct, Genome Wide, CRISPR, Functional Assay, Binding Assay, Analysis

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Influence of adrenomedullin on the enzymatic activity of dipeptidyl peptidase 3 in vitro

doi: 10.1080/14756366.2026.2700037

Figure Lengend Snippet: Antibody-mediated reversal of peptide-induced inhibition of DPP3 activity. ADM (panel A), ADM1–14 (panel B), and ADM-Gly (panel C), respectively, was added at a constant final concentration of 10 µM in the DPP3 activity assay, and increasing amounts of the anti-N-terminal ADM antibody and the negative control antibody Palivizumab, respectively, were added at the indicated concentrations. The inhibition achieved with 10 µM of each peptide was defined as 100% inhibition, and inhibition measured in the presence of antibodies was related to this. The maximal reversal of peptide-induced inhibition of DPP3 activity was defined as 0% inhibition. Shown are mean (SD) values gained from duplicate measurements. For all three peptides, the reversal of peptide-induced inhibition of DPP3 activity by the anti-ADM antibody was statistically significant compared to the negative control antibody Palivizumab ( p > 0.05 by ANOVA).

Article Snippet: A humanised anti-N-terminal ADM monoclonal antibody (initially named Adrecizumab, later Enibarcimab) was purchased from (MedChemExpress, Monmouth Junction, USA).

Techniques: Inhibition, Activity Assay, Concentration Assay, Negative Control