Journal: Advanced Science

Article Title: GOT1 Inhibition Induces Extracellular Matrix Remodeling in Pancreatic Cancer

doi: 10.1002/advs.202516578

Figure Lengend Snippet: Effects of GOT1 inhibition in the pancreatic TME. (a) Schematic of glutamine metabolism. (b) Determination of half‐maximal inhibitory concentration (IC 50 ) of the GOT1 inhibitor using mono‐ and multicellular 3D cultures. (c) Cell viability of mono‐ and multicellular 3D cultures. n = 3, **** = p ≤ 0.0001, (d) live/dead staining (scale bar = 100 µm), and (e) relative metabolite quantification following GOT1 inhibition. (f) Gene ontology enrichment analysis, and (g) volcano plot showing the significantly up/downregulated matrisome proteins of treated versus non‐treated multicellular 3D cultures. Blue = upregulated, red = downregulated. FDR cut‐off = 0.05, log2(fold‐change) ≥ 2. (h) Immunoblot of multicellular 3D cultures upon GOT1 inhibition. (i) Cell metabolic activity for PDAC cells cultured in type III collagen. n = 6, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001. (j) Cytokine profiling of multicellular 3D cultures following GOT1 inhibition. GOT1, glutamic‐oxaloacetic transaminase 1. TME, tumor microenvironment. PDAC, pancreatic ductal adenocarcinoma.

Article Snippet: Mono‐ and multicellular 3D cultures were established as described above and grown for 7 days and then treated with 25 μM GOT1 inhibitor (MedchemExpress HY‐122723) alone or combined with 100 nM gemcitabine (Sigma‐Aldrich G6423) and 100 nM nab‐paclitaxel (Bristol‐Myers Squibb), which were dissolved directly into PDAC or TKCC cell media.

Techniques: Inhibition, Concentration Assay, Staining, Western Blot, Activity Assay, Cell Culture

Journal: Advanced Science

Article Title: GOT1 Inhibition Induces Extracellular Matrix Remodeling in Pancreatic Cancer

doi: 10.1002/advs.202516578

Figure Lengend Snippet: Analyses of cell responses following combined GOT1 inhibition and treatment with cytotoxic drugs. (a) Schematic of experimental rationale. (b) Cell viability of mono‐ and multicellular 3D cultures grown in GelMA‐HAMA hydrogels following treatment with GOT1 inhibitor ± gemcitabine and nab‐paclitaxel. n = 3, * = p ≤ 0.05, *** = p ≤ 0.001. (c) Gene ontology enrichment analysis, (d) signaling pathway analysis, and (e) volcano plot showing significantly up/downregulated matrisome proteins of treated multicellular 3D cultures compared to non‐treated cells grown in GelMA‐HAMA hydrogels. Blue = upregulated, red = downregulated. (f) Relative metabolite quantification in multicellular 3D cultures following treatment with GOT1 inhibitor ± gemcitabine and nab‐paclitaxel. FDR cut‐off = 0.05, log2(fold‐change) ≥ 2. GOT1, glutamic‐oxaloacetic transaminase 1. GelMA, gelatin methacrylate. HAMA, hyaluronic acid methacrylate.

Article Snippet: Mono‐ and multicellular 3D cultures were established as described above and grown for 7 days and then treated with 25 μM GOT1 inhibitor (MedchemExpress HY‐122723) alone or combined with 100 nM gemcitabine (Sigma‐Aldrich G6423) and 100 nM nab‐paclitaxel (Bristol‐Myers Squibb), which were dissolved directly into PDAC or TKCC cell media.

Techniques: Inhibition

Journal: Advanced Science

Article Title: GOT1 Inhibition Induces Extracellular Matrix Remodeling in Pancreatic Cancer

doi: 10.1002/advs.202516578

Figure Lengend Snippet: Multicellular 3D cultures including patient‐derived PDAC cells and their response toward GOT1 inhibition. (a) Micrograph of multicellular 3D cultures grown in GelMA‐HAMA hydrogels (scale bar = 100 µm). (b) Cell metabolic activity and (c) DNA content of mono‐ and multicellular 3D cultures including patient‐derived PDAC cells in GelMA‐HAMA hydrogels. (d) Cell viability, and (e) DNA content of mono‐ and multicellular TKCC‐22‐LO cultures following treatment with GOT1 inhibitor ± gemcitabine and nab‐paclitaxel. n = 3, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001. (f) Live/dead staining (scale bar = 100 µm), and (g) heatmap of amino acid metabolism microarray of multicellular 3D cultures treated with the GOT1 inhibitor compared to non‐treated cells. PDAC, pancreatic ductal adenocarcinoma. GOT1, glutamic‐oxaloacetic transaminase 1. GelMA, gelatin methacrylate. HAMA, hyaluronic acid methacrylate.

Article Snippet: Mono‐ and multicellular 3D cultures were established as described above and grown for 7 days and then treated with 25 μM GOT1 inhibitor (MedchemExpress HY‐122723) alone or combined with 100 nM gemcitabine (Sigma‐Aldrich G6423) and 100 nM nab‐paclitaxel (Bristol‐Myers Squibb), which were dissolved directly into PDAC or TKCC cell media.

Techniques: Derivative Assay, Inhibition, Activity Assay, Staining, Microarray

Journal: Journal of Virology

Article Title: African swine fever virus hijacks host pyrimidine metabolism to promote viral replication

doi: 10.1128/jvi.00985-25

Figure Lengend Snippet: Endogenous aspartate synthesis regulates ASFV replication. ( A ) Exogenous aspartate deficiency does not affect the replication of ASFV (normal culture medium containing 150 µM aspartate). ( B ) Schematic diagram of endogenous synthesis pathway of aspartate. ( C ) Detection of the effect of aminooxyacetic acid hemihydrochloride (AOA) on the expression of ASFV-P30 protein using Western blotting. ( D ) RT-qPCR analysis of ASFV-B646L mRNA expression in infected cells treated with GOT1 siRNA. ( E ) Western blot and virus titration assessing the effect of GOT1 siRNA on ASFV-P30 expression and infectious virus production. ( F ) Exogenous aspartate supplementation restores ASFV-P30 protein levels suppressed by GOT1 siRNA. ( G ) RT-qPCR analysis of ASFV-B646L mRNA expression in infected cells treated with GOT2 siRNA. ( H ) Detection of the effect of GOT2 siRNA on the expression of ASFV-P30 protein expression and virus titer. For all experiments, PAMs were infected with ASFV at MOI = 1, and cell lysates were collected at 24 hpi for Western blotting. Statistical significance is indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. = no significant difference ( P ≥ 0.05).

Article Snippet: The antibodies used in this study included ASFV-P30 mouse monoclonal antibody (produced by our laboratory), β-tubulin ( M20005 ; Abmart), GOT1 (14886-1-AP; Proteintech), and GOT2 (67738-1-Ig; Proteintech).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Infection, Virus, Titration

Journal: Journal of Virology

Article Title: African swine fever virus hijacks host pyrimidine metabolism to promote viral replication

doi: 10.1128/jvi.00985-25

Figure Lengend Snippet: Temporal GOT1–GOT2 reciprocity directs aspartate flux for ASFV replication. ( A, B ) Expression levels of GOT1 and GOT2 during ASFV infection at various time points. ( C, D ) RT-qPCR and Western blot analysis of changes in GOT1 expression after GOT2 knockdown by siRNA. ( E ) Exogenous nucleoside supplementation restores ASFV-P30 protein levels suppressed by GOT1 siRNA. Unless otherwise indicated, PAMs were infected with ASFV at MOI = 1, and samples were collected at 24 hpi for Western blotting, RT-qPCR, and viral titer analysis. Statistical significance is indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, n.s. = no significant difference ( P ≥ 0.05).

Article Snippet: The antibodies used in this study included ASFV-P30 mouse monoclonal antibody (produced by our laboratory), β-tubulin ( M20005 ; Abmart), GOT1 (14886-1-AP; Proteintech), and GOT2 (67738-1-Ig; Proteintech).

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Knockdown

Journal: Journal of Virology

Article Title: African swine fever virus hijacks host pyrimidine metabolism to promote viral replication

doi: 10.1128/jvi.00985-25

Figure Lengend Snippet: Schematic diagram illustrating the mechanisms by which ASFV regulates nucleotide synthesis precursors through multiple pathways. ASFV reprograms host central carbon and nitrogen metabolism to fuel de novo pyrimidine nucleotide synthesis. First, ASFV activates the PPP, diverting glucose-derived flux to generate R5P, which is essential for nucleotide backbone synthesis. Second, ASFV upregulates the expression of the glutamine transporter SLC1A5, enhancing cellular glutamine uptake. Imported glutamine serves dual roles: (i) as a nitrogen donor for nucleotide biosynthesis, and (ii) as a carbon source, being converted to α-KG via glutaminolysis. α-KG enters the TCA cycle and is further converted by GOT1 to produce aspartate, which is required for pyrimidine ring formation. Together, these pathways ensure an adequate supply of nucleotide precursors to support ASFV DNA replication and gene expression. Viral hijacking of host metabolism thus represents a coordinated strategy to sustain robust viral replication.

Article Snippet: The antibodies used in this study included ASFV-P30 mouse monoclonal antibody (produced by our laboratory), β-tubulin ( M20005 ; Abmart), GOT1 (14886-1-AP; Proteintech), and GOT2 (67738-1-Ig; Proteintech).

Techniques: Derivative Assay, Expressing, Gene Expression