Journal: Cell Death Discovery

Article Title: Glycolytic reprogramming mediated by the ADAM12/IGF1 axis promotes ossification of the posterior longitudinal ligament

doi: 10.1038/s41420-026-03044-8

Figure Lengend Snippet: A Representative immunofluorescence images and quantification of glycolytic enzymes PKM2 and LDHA in human PLL and OPLL tissues. Scale bar = 100 µm, n = 3. B Western blot analysis of GLUT1, PKM2, and LDHA protein levels in PLL and OPLL cells. n = 3. C , D . Glucose uptake and lactate production in PLL and OPLL cells. n = 3. E ECAR measured by Seahorse XF Analyzer to assess the glycolytic capacity of PLL and OPLL cells. n = 3. F Representative images showing TMRE staining (orange-red) in PLL and OPLL cells. The bar graph depicts the quantification of relative TMRE fluorescence intensity. Scale bar = 30 µm, n = 3. G Western blot analysis of PDK1, Cyto c, and ATP5A in OPLL and PLL cells. H Intracellular ROS levels measured by DCFH-DA fluorescence. Scale bar = 50 µm. I OCR measured by Seahorse XF Analyzer to assess mitochondrial respiration. n = 3. J Western blot analysis of RUNX2, OSX, and ALP during a 15day time course of osteogenic differentiation of ligament cells. K Western blot analysis of GLUT1, HK2, and LDHA during a 15day time course of osteogenic differentiation of ligament cells. L Western blot analysis of PDK1, Cyto c, and ATP5A during a 15day time course of osteogenic differentiation of ligament cells. Data are presented as mean ± standard deviation.

Article Snippet: The primary antibodies and dilutions used were: ADAM12 (Proteintech, 14139-1-AP, 1:100), RUNX2 (Abclonal, A11753, 1:600), OCN (Proteintech, 23418-1-IG, 1:600), PKM2 (Proteintech, 60268-1-IG, 1:1500), LDHA (Servicebio, GB11342-100, 1:300), CD31 (Servicebio, GB11063-2-50, 1:1500), PRG4 (Abcam, ab28484, 1:200) and Vimentin (Proteintech, 10366-1-AP, 1:200).

Techniques: Immunofluorescence, Western Blot, Staining, Fluorescence, Standard Deviation

Journal: Cell Death Discovery

Article Title: Glycolytic reprogramming mediated by the ADAM12/IGF1 axis promotes ossification of the posterior longitudinal ligament

doi: 10.1038/s41420-026-03044-8

Figure Lengend Snippet: A Venn diagram illustrating the overlap of hub genes identified from the PPI network using five different centrality algorithms: MCC, DMNC, EPC, MNC, and Degree. B Key genes identified by the DMNC method within the PPI network. C Correlation analysis between the glycolysis flux score and ADAM12 expression in OPLL and PLL tissues. D Distribution of ADAM12 across the major cell types in posterior longitudinal ligament tissue. E Scatter plot showing the positive correlation between ADAM12 expression and the glucose metabolism pathway activity score across individual ligament cells. F Representative immunofluorescence images and quantification of ADAM12 expression in human OPLL and PLL tissues. Scale bar = 50 μm, n = 3. G Representative immunofluorescence images showing co-localization of ADAM12 (green) and PKM2 (red) in OPLL tissue. Scale bar = 30 μm. H , I Quantitative PCR analysis of ADAM12L and ADAM12S mRNA levels in PLL and OPLL cells. n = 3. J Quantitative PCR analysis of the expression levels of the ADAM12L and ADAM12S isoforms in OPLL cells. n = 3. K Western blot analysis of ADAM12 protein levels in PLL and OPLL cells. n = 3. L ELISA analysis of ADAM12 protein levels in PLL and OPLL ligament cells supernatant. n = 5. Data are presented as mean ± standard deviation.

Article Snippet: The primary antibodies and dilutions used were: ADAM12 (Proteintech, 14139-1-AP, 1:100), RUNX2 (Abclonal, A11753, 1:600), OCN (Proteintech, 23418-1-IG, 1:600), PKM2 (Proteintech, 60268-1-IG, 1:1500), LDHA (Servicebio, GB11342-100, 1:300), CD31 (Servicebio, GB11063-2-50, 1:1500), PRG4 (Abcam, ab28484, 1:200) and Vimentin (Proteintech, 10366-1-AP, 1:200).

Techniques: Expressing, Activity Assay, Immunofluorescence, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Cell Death Discovery

Article Title: Glycolytic reprogramming mediated by the ADAM12/IGF1 axis promotes ossification of the posterior longitudinal ligament

doi: 10.1038/s41420-026-03044-8

Figure Lengend Snippet: A Heatmap showing the relative abundance of central carbon metabolites in control (NC), osteogenically differentiated (NC-OD), and ADAM12-overexpressing osteogenically differentiated (OE-OD) ligament cells. n = 3. B Box plots quantifying the levels of key glycolytic intermediates (fructose-1,6-bisphosphate, 3-phosphoglycerate, pyruvate, and lactate) from the metabolomics data. n = 3. C , D Western blot analysis of glycolytic markers (GLUT1, HK2, PKM2, LDHA) and mitochondrial markers (PGC1α, mtTFA, ATP5A, cytochrome c) in ligament cells after ADAM12 knockdown and overexpression. n = 3. E , F Glucose uptake capacity in ligament cells after ADAM12 knockdown and overexpression. n = 3. G , H Lactate production in ligament cells after ADAM12 knockdown and overexpression. n = 3. I , J Intracellular pyruvate content in ligament cells after ADAM12 knockdown and overexpression. n = 3. K , L ECAR measured by Seahorse XF Analyzer in ligament cells after ADAM12 knockdown and overexpression. n = 3. Data are presented as mean ± standard deviation.

Article Snippet: The primary antibodies and dilutions used were: ADAM12 (Proteintech, 14139-1-AP, 1:100), RUNX2 (Abclonal, A11753, 1:600), OCN (Proteintech, 23418-1-IG, 1:600), PKM2 (Proteintech, 60268-1-IG, 1:1500), LDHA (Servicebio, GB11342-100, 1:300), CD31 (Servicebio, GB11063-2-50, 1:1500), PRG4 (Abcam, ab28484, 1:200) and Vimentin (Proteintech, 10366-1-AP, 1:200).

Techniques: Control, Western Blot, Knockdown, Over Expression, Standard Deviation

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 for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

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. 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.

Article Snippet: Primary antibodies used for CUT7RUN in this study includes: CDK8 (ProteinTech, #22067), SIX4 (Santa Cruz Biotechnology, #SC-390779), HA (Cell Signaling Technology, #C29F4-3724), TADA2B (ProteinTech, #17367), CCNC (ProteinTech, #26464), MED13 (ProteinTech, #26464), MED12 (ProteinTech, #20028), H3K4me3 (EpiCypher, #13-0041), H3K27ac (Cell Signaling Technology, #8173S).

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