Journal: Tumour Virus Research

Article Title: Oncolytic virus hijacks GOT1 and pyrimidinosomes to fuel pyrimidine synthesis for replication in tumor cells

doi: 10.1016/j.tvr.2026.200342

Figure Lengend Snippet: | NDV infection promotes the assembly of pyrimidinosome involving GOT1 and pyrimidine synthases. (A) H1299 cells were co-transfected with GFP-GOT1 and mCherry-UMPS for 24 h, followed by mock infection or NDV infection (MOI = 0.1, 1, 5, 10, or UV-NDV for 12 hpi). Cells were then analyzed using confocal microscopy. (B) H1299 cells were co-transfected with GFP-GOT1 and mCherry-UMPS for 24 h, followed by mock infection or NDV infection (MOI = 1 for 6, 12, and 18 hpi). Cells were then analyzed using confocal microscopy. (C) Immunofluorescence analysis (IFA) of mock-infected and NDV-infected A549 cells (MOI = 1, 12 hpi), stained for endogenous GOT1, UMPS, and DHODH, alongside the mitochondrial marker protein Tom 20. (D-F) H1299 cells were co-transfected with GFP-GOT1 and mCherry-UMPS (D), GFP-DHODH and mCherry-UMPS (E), or GFP-GOT1 and mCherry-DHODH (F) for 24 h, followed by mock infection or NDV infection (MOI = 1, 12 hpi). Cells were then analyzed using confocal microscopy. (G-H) HEK293T cells were mock-infected or infected with NDV (MOI = 1) for 12 h. The cells were lysed and subjected to immunoprecipitation (IP) using GOT1 (G) and UMPS (H) antibodies, alongside with anti-IgG antibody as a control, followed by WB with anti-CAD, -UMPS, -DHODH and -GOT1 antibodies. (I-J) H1299 cells were co-transfected with Flag-CAD and HA-GOT1 for 24 h, followed by mock infection or NDV infection (MOI = 1, 12 hpi). The cells were lysed and subjected to IP using anti-Flag (I) or anti-HA (J) magnetic beads, followed by WB with anti-Flag and anti-HA antibodies. (K) H1299 cells were transfected with GFP-GOT1, mCherry-UMPS, or GFP-DHODH for 24 h, followed by NDV infection (MOI = 1, 12 hpi). FRAP analysis was then performed using confocal microscopy.

Article Snippet: Anti-Flag Magnetic Beads (Cat# HY-K0207), anti-HA Magnetic Beads (Cat# HY-K0201), Mycophenolate (Cat# HY-B0421), Leflunomide (Cat# HY-B0083), AG 2037 (Cat# HY-14530), Aminooxyacetic acid hemihydrochloride (Cat# HY-107994) were purchased from MedChemExpress (MCE).

Techniques: Infection, Transfection, Confocal Microscopy, Immunofluorescence, Staining, Marker, Immunoprecipitation, Control, Magnetic Beads