Journal: The Journal of Biological Chemistry

Article Title: Cardiolipin-induced activation of pyruvate dehydrogenase links mitochondrial lipid biosynthesis to TCA cycle function

doi: 10.1074/jbc.RA119.009037

Figure Lengend Snippet: Decreased activity of PDH in tafazzin-deficient cells. A, PDH activity was assayed in isolated mitochondria as described under “Experimental procedures.” Data shown are mean ± S.D. (n = 3). *, p < 0.05. B, levels of PDH-E1α in mitochondrial extracts were determined by Western blot analysis with anti-PDH-E1α antibody. 50 μg of total protein from each sample was loaded onto an SDS gel under reducing conditions, and the mitochondrial protein Ndufb6 was used as a loading control. C, PDH activity was assayed in heart tissue extracted from WT and TAZ-KO mice. Each column represents a separate mouse. Data shown are mean ± S.D. (n = 2). D, WT and TAZ-KO cells were seeded in DMEM (without glucose, pyruvate, or glutamine/1% FCS), followed by 3-h incubation in Dulbecco's PBS (Life Technologies) supplemented with 100 μm [1-14C]pyruvate, and the PDH flux assay was performed in the presence or absence of 5 mm DCA. PDH activity was assayed by measuring the release of 14CO2 from [1-14C]pyruvate as described under “Experimental procedures.” Data shown are mean ± S.D. (n = 3). *, p < 0.05.

Article Snippet: PDH bound to lipids on the membrane was detected by immunoblotting using primary antibody (Assaypro) against the PDH complex (1:1000) and secondary antibody conjugated to horseradish peroxidase (1:3000).

Techniques: Activity Assay, Isolation, Western Blot, SDS-Gel, Incubation, Flux Assay

Journal: The Journal of Biological Chemistry

Article Title: Cardiolipin-induced activation of pyruvate dehydrogenase links mitochondrial lipid biosynthesis to TCA cycle function

doi: 10.1074/jbc.RA119.009037

Figure Lengend Snippet: CL binds to PDH and increases its activity. A, digitonin-solubilized mitochondria from WT and TAZ-KO cells were treated with or without CL. PDH activity was assayed as described under “Experimental procedures.” B, purified PDH complex (Sigma) was incubated with exogenous CL or phosphatidic acid (PA). PDH activity was assayed as described under “Experimental procedures”. Data shown are mean ± S.D. (n = 3). *, p < 0.05. C, the indicated lipids CL, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 3,4,5-trisphosphate (PIP3), and phosphatidic acid were serially diluted and spotted onto a nitrocellulose membrane, which was incubated overnight in buffer containing 10 μg of PDH complex. Interactions were detected by immunoblotting with an antibody against the PDH complex.

Article Snippet: PDH bound to lipids on the membrane was detected by immunoblotting using primary antibody (Assaypro) against the PDH complex (1:1000) and secondary antibody conjugated to horseradish peroxidase (1:3000).

Techniques: Activity Assay, Purification, Incubation, Western Blot

Journal: NPJ Precision Oncology

Article Title: Cuproptosis-associated PDHA1 promotes sarcoma progression and immunotherapy responsiveness via the E2F1–PD-L1 axis: a multi-omics and clinical validation study

doi: 10.1038/s41698-026-01298-0

Figure Lengend Snippet: Overview of the study revealing PDHA1 as a key regulator of sarcoma progression and immune evasion.

Article Snippet: To modulate cuproptosis, MG63 cells with stable PDHA1 knockdown or control (sh-PDHA1 and sh-NC) were treated with the copper chelator ammonium tetrathiomolybdate (TTM; MedChemExpress, HY-100276).

Techniques:

Journal: NPJ Precision Oncology

Article Title: Cuproptosis-associated PDHA1 promotes sarcoma progression and immunotherapy responsiveness via the E2F1–PD-L1 axis: a multi-omics and clinical validation study

doi: 10.1038/s41698-026-01298-0

Figure Lengend Snippet: A Cumulative distribution function (CDF) plots for consensus clustering with k = 2–6. B Relative change in the area under the CDF curve for different k-values. C Consensus matrix for clustering at k = 2. D Heatmap of CRG expression across subtypes. E Differential expression of CRGs between C1 and C2 in TCGA-SARC. F Protein-protein interaction network of 12 CRGs, highlighting PDHA1 as a hub. G Pearson correlation matrix of CRG expression. H Friends similarity analysis showing PDHA1 as the most central gene.

Article Snippet: To modulate cuproptosis, MG63 cells with stable PDHA1 knockdown or control (sh-PDHA1 and sh-NC) were treated with the copper chelator ammonium tetrathiomolybdate (TTM; MedChemExpress, HY-100276).

Techniques: Expressing, Quantitative Proteomics

Journal: NPJ Precision Oncology

Article Title: Cuproptosis-associated PDHA1 promotes sarcoma progression and immunotherapy responsiveness via the E2F1–PD-L1 axis: a multi-omics and clinical validation study

doi: 10.1038/s41698-026-01298-0

Figure Lengend Snippet: A PDHA1 expression across GEO, TARGET, and ICGC. B ROC curves for PDHA1 diagnostic value. C Distribution of risk score, survival status, and expression in TCGA. D Kaplan-Meier curve of OS. E Time-dependent ROC curves for 1-, 3-, and 5-year OS. F DSS curve of PDHA1 high and low groups. G OS curve in GSE21257 . H OS curve in GSE17674 .

Article Snippet: To modulate cuproptosis, MG63 cells with stable PDHA1 knockdown or control (sh-PDHA1 and sh-NC) were treated with the copper chelator ammonium tetrathiomolybdate (TTM; MedChemExpress, HY-100276).

Techniques: Expressing, Diagnostic Assay

Journal: NPJ Precision Oncology

Article Title: Cuproptosis-associated PDHA1 promotes sarcoma progression and immunotherapy responsiveness via the E2F1–PD-L1 axis: a multi-omics and clinical validation study

doi: 10.1038/s41698-026-01298-0

Figure Lengend Snippet: A Nomogram integrating PDHA1 and clinical variables. B Calibration curves for 1-, 3-, and 5-year OS. C ROC curves for OS prediction. D Time-dependent AUC curves. E DCA showing clinical benefit of the nomogram.

Article Snippet: To modulate cuproptosis, MG63 cells with stable PDHA1 knockdown or control (sh-PDHA1 and sh-NC) were treated with the copper chelator ammonium tetrathiomolybdate (TTM; MedChemExpress, HY-100276).

Techniques:

Journal: NPJ Precision Oncology

Article Title: Cuproptosis-associated PDHA1 promotes sarcoma progression and immunotherapy responsiveness via the E2F1–PD-L1 axis: a multi-omics and clinical validation study

doi: 10.1038/s41698-026-01298-0

Figure Lengend Snippet: A Correlation between PDHA1 expression levels and infiltration of various immune cells across 33 different tumor types, analyzed using CIBERSORT and ssGSEA algorithms (* P < 0.05). B Differences in immune infiltration between high and low PDHA1 expression groups based on the CIBERSORT algorithm. C Correlation analysis between PDHA1 expression and immune infiltration based on the CIBERSORT algorithm. D Differences in immune infiltration between high and low PDHA1 expression groups based on the ssGSEA algorithm. E Correlation analysis between PDHA1 expression and immune infiltration based on the ssGSEA algorithm. F Differences in ESTIMATE scores between high and low PDHA1 expression groups.

Article Snippet: To modulate cuproptosis, MG63 cells with stable PDHA1 knockdown or control (sh-PDHA1 and sh-NC) were treated with the copper chelator ammonium tetrathiomolybdate (TTM; MedChemExpress, HY-100276).

Techniques: Expressing

Journal: NPJ Precision Oncology

Article Title: Cuproptosis-associated PDHA1 promotes sarcoma progression and immunotherapy responsiveness via the E2F1–PD-L1 axis: a multi-omics and clinical validation study

doi: 10.1038/s41698-026-01298-0

Figure Lengend Snippet: A scRNA-seq clustering of SARC immune cells. B Cell lineage annotations. C Heatmap of gene expression aggregated by cell type. D Dot plot of canonical marker genes. E PDHA1 distribution across cell types. F Cell–cell communication network. G Ligand–receptor interaction bubble plot.

Article Snippet: To modulate cuproptosis, MG63 cells with stable PDHA1 knockdown or control (sh-PDHA1 and sh-NC) were treated with the copper chelator ammonium tetrathiomolybdate (TTM; MedChemExpress, HY-100276).

Techniques: Gene Expression, Marker

Journal: NPJ Precision Oncology

Article Title: Cuproptosis-associated PDHA1 promotes sarcoma progression and immunotherapy responsiveness via the E2F1–PD-L1 axis: a multi-omics and clinical validation study

doi: 10.1038/s41698-026-01298-0

Figure Lengend Snippet: A PDHA1 mRNA levels are significantly higher in sarcoma tissues than in matched adjacent normal tissues ( n = 60). B , C Western blot and quantitative analysis showing increased protein expression of PDHA1, PD-L1 and E2F1 in tumor versus normal tissues. D , E Representative IHC staining and semiquantitative analysis in liposarcoma and synovial sarcoma samples demonstrating upregulation of PDHA1, PD-L1, E2F1 and Ki-67 in tumor tissues. Scale bar: 50 μm. F Nomogram incorporating PDHA1 expression, age, gender and metastasis status to predict 1-, 3- and 5-year overall survival (OS) in sarcoma patients. G Kaplan–Meier analysis indicating that high PDHA1 expression is associated with poor OS. H Time-dependent ROC curves showing the predictive performance of the PDHA1-based model for 1-, 3- and 5-year OS. I Dynamic AUC plot evaluating the discrimination ability of the nomogram over time. J Decision curve analysis (DCA) demonstrating the net clinical benefit of the PDHA1-based nomogram. K – M qPCR and Western blot analyses showing that PDHA1 expression is significantly elevated in human sarcoma cell lines (MG63, SW982, SW872) compared with normal control cells (hFOB1.19, HFLS, HPA-V). Data are presented as mean ± SEM. *** P < 0.001.

Article Snippet: To modulate cuproptosis, MG63 cells with stable PDHA1 knockdown or control (sh-PDHA1 and sh-NC) were treated with the copper chelator ammonium tetrathiomolybdate (TTM; MedChemExpress, HY-100276).

Techniques: Western Blot, Expressing, Immunohistochemistry, Control

Journal: NPJ Precision Oncology

Article Title: Cuproptosis-associated PDHA1 promotes sarcoma progression and immunotherapy responsiveness via the E2F1–PD-L1 axis: a multi-omics and clinical validation study

doi: 10.1038/s41698-026-01298-0

Figure Lengend Snippet: A qRT-PCR analysis showing efficient knockdown of PDHA1 by three independent shRNAs and corresponding downregulation of PD-L1 and E2F1 in MG63 and SW982 cells. B Western blot confirms reduced protein levels of PDHA1, E2F1, and PD-L1 in both cell lines. C , D CCK-8 assays demonstrate significantly impaired proliferation after PDHA1 silencing at 24–72 h. E , F EdU immunofluorescence staining and quantification show decreased DNA synthesis in PDHA1-deficient cells. G , H Wound healing assays reveal reduced migration capacity after PDHA1 knockdown. I , J Transwell migration and invasion assays indicate markedly decreased motility and invasiveness in both MG63 and SW982 cells. K , L Colony formation assays demonstrate a substantial reduction in clonogenic potential upon PDHA1 depletion.

Article Snippet: To modulate cuproptosis, MG63 cells with stable PDHA1 knockdown or control (sh-PDHA1 and sh-NC) were treated with the copper chelator ammonium tetrathiomolybdate (TTM; MedChemExpress, HY-100276).

Techniques: Quantitative RT-PCR, Knockdown, Western Blot, CCK-8 Assay, Immunofluorescence, Staining, DNA Synthesis, Migration

Journal: NPJ Precision Oncology

Article Title: Cuproptosis-associated PDHA1 promotes sarcoma progression and immunotherapy responsiveness via the E2F1–PD-L1 axis: a multi-omics and clinical validation study

doi: 10.1038/s41698-026-01298-0

Figure Lengend Snippet: A – C 3D spheroid assays in MG63 cells show significantly reduced spheroid volume and Matrigel invasion area following PDHA1 knockdown over 7 days. D – F In vivo xenograft models demonstrate markedly suppressed tumor growth and decreased tumor weight in the sh-PDHA1 group. G Representative H&E, TUNEL, and IHC staining of xenograft tumors show decreased expression of PDHA1, Ki-67, Cyclin D1, PD-L1, E2F1, Bcl-2, and MMP-9, and increased cleaved Caspase-3 and apoptosis in the sh-PDHA1 group. H Quantification of TUNEL and IHC staining. I , J Western blot and densitometric analysis confirm significant downregulation of PDHA1, PD-L1, E2F1, Bcl-2, and MMP-9, and upregulation of cleaved Caspase-3 in tumor lysates upon PDHA1 knockdown.

Article Snippet: To modulate cuproptosis, MG63 cells with stable PDHA1 knockdown or control (sh-PDHA1 and sh-NC) were treated with the copper chelator ammonium tetrathiomolybdate (TTM; MedChemExpress, HY-100276).

Techniques: Knockdown, In Vivo, TUNEL Assay, Immunohistochemistry, Expressing, Western Blot

Journal: NPJ Precision Oncology

Article Title: Cuproptosis-associated PDHA1 promotes sarcoma progression and immunotherapy responsiveness via the E2F1–PD-L1 axis: a multi-omics and clinical validation study

doi: 10.1038/s41698-026-01298-0

Figure Lengend Snippet: A ChIP-qPCR analysis showing enrichment of E2F1 at the predicted response elements (RE1, RE2) in the PD-L1 promoter in MG63 and SW982 cells; enrichment is further increased upon E2F1 overexpression (Ac-H3, positive control; IgG, negative control). B , C Luciferase assays indicating that E2F1 enhances the activity of wild-type (WT) PD-L1 promoter constructs but not mutant (Mut) constructs lacking E2F1-binding sites. D , E Western blot and quantification of PDHA1, E2F1, and PD-L1 in sh-NC, sh-PDHA1, sh-PDHA1 + E2F1-OE, and E2F1-OE groups, showing that E2F1 rescues PD-L1 downregulation induced by PDHA1 knockdown. F , G Flow cytometry and statistical analysis of apoptosis demonstrating that PDHA1 knockdown increases apoptosis, which is partially reversed by E2F1 overexpression. H , I Western blot and quantification showing that PDHA1 knockdown and/or TTM treatment reduce Lip-DLAT, E2F1, and PD-L1 expression, with the strongest inhibition in the combination group. J , K Multiplex immunofluorescence and quantification in sarcoma tissues showing that high PDHA1 expression is associated with higher PD-L1 and reduced CD8⁺ T-cell infiltration. Scale bars: 50 μm. L Schematic model of the PDHA1–E2F1–PD-L1 axis in mediating CD8⁺ T-cell exclusion and immune evasion. Data are shown as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To modulate cuproptosis, MG63 cells with stable PDHA1 knockdown or control (sh-PDHA1 and sh-NC) were treated with the copper chelator ammonium tetrathiomolybdate (TTM; MedChemExpress, HY-100276).

Techniques: ChIP-qPCR, Over Expression, Positive Control, Negative Control, Luciferase, Activity Assay, Construct, Mutagenesis, Binding Assay, Western Blot, Knockdown, Flow Cytometry, Expressing, Inhibition, Multiplex Assay, Immunofluorescence