Journal: bioRxiv

Article Title: The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation

doi: 10.64898/2026.04.03.716411

Figure Lengend Snippet: (A) A schematic illustrates the activation of STING, TBK1, and IRF3 in response to double-stranded DNA (dsDNA) in the cytosol by cGAS. (B-D) THP-1 macrophages were mock-infected or infected with HSV-1 in the presence or absence of 1 µM PGE 2 . At the indicated h.p.i, cell lysates were collected and subjected to immunoblotting with the indicated antibodies (B) . Band intensity of phosphorylated TBK1 (C) and IRF3 (D) was quantified and normalized to total TBK1 and IRF3, respectively (n = 3). (E) THP-1 macrophages expressing ISRE-Luciferase were transfected with viral DNA at indicated concentrations in the presence or absence of 1 µM PGE 2 for 16 hours. Cell lysates were collected and subjected to a luciferase reporter assay to assess ISRE promoter activity (n = 3). (F) THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1) for the indicated times. Cell lysates were collected and subjected to immunoblotting with the indicated antibodies. Samples transfected with either scramble siRNA (siCTRL) or TFAM siRNA (siTFAM) were included as controls. (G) THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1). At 16 h.p.i, cell lysates were collected and subjected to qPCR to assess levels of total mt-D-loop and mt-ND1 regions (n = 3). (H) Representative Airyscan live-cell imaging of THP-1 macrophages mock-infected or infected with HSV-1 for 3 hours. MitoTracker DeepRed (magenta): mitochondria, PicoGreen (yellow): DNA. Scale bars, 2 µm. (I) Quantification of cytosolic PicoGreen in THP-1 macrophages mock-infected or infected with HSV-1 (MOI = 1) for 3 hours (n = 10). (J-K) THP-1 macrophages were infected with HSV-1 in the presence or absence of the mtDNA replication inhibitor, ddC. At 24 h.p.i, cell supernatants were collected and subjected to ELISA to measure levels of secreted IFNβ (n = 3) (J) , and cell lysates were collected and subjected to qPCR to quantify HSV-1 UL30 genomic abundance (n = 3) (K) . (L) THP-1 macrophages expressing ISRE-Luciferase were transfected with either scramble siRNA (siCTRL) or TFAM siRNA (siTFAM) in the presence or absence of 1 µM PGE 2. At 16 hours post stimulation, cell lysates were collected and subjected to a luciferase reporter assay to assess ISRE promoter activity (n = 3). (M) THP-1 macrophages were transfected with either scramble siRNA (siCTRL) or TFAM siRNA (siTFAM) in the presence or absence of 1 µM PGE 2 . At 16 hours post-stimulation, cell lysates were collected and subjected to RT-qPCR to assess the mRNA levels of representative type I ISGs (n = 3). All experiments were performed with three independent biological replicates and repeated at least twice with reproducible results. Data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test ( C-E, L-M ) or unpaired, two-tailed Student’s t-test ( G, I-K ). p -values are indicated.

Article Snippet: Antibodies for PKA substrates (#9624), PKA Cα (#5842), PKA RIα/β (#3927), GAPDH (#2118), STING (#13647), pTBK1 S172 (5483), TBK1 (#3504), pIRF3 S396 (#4947), IRF3 (#4302), TFAM (#8076), HSP90 (#4877), pUB S65 (#62802), COX4 (#4850), pPINK1 S228 (#89010), STOML2 (#89199), Vinculin (#13901) were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Infection, Western Blot, Expressing, Luciferase, Transfection, Reporter Assay, Activity Assay, Live Cell Imaging, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Two Tailed Test