p stat2 y690  (Cell Signaling Technology Inc)

Journal: Pathogens

Article Title: Physalin F Promotes AFG3L2-Mediated Degradation of VISA/MAVS to Suppress Innate Immune Response to RNA Virus

doi: 10.3390/pathogens15010074

Figure Lengend Snippet: Identification of physalin F as an inhibitor of SeV-induced innate immune signaling. ( A ) Compounds of a natural small molecule library ( n = 903) were sub-pooled into 4 compounds/sub-pool. HEK293T cells (5 × 10 4 ) stably transduced with an ISRE luciferase reporter were treated with each of the sub-pools (5 μM for each compound) for 0.5 h, followed by infection with SeV for 8 h before luciferase assays (left dot plot, the dashed line indicates inhibition of SeV-induced ISRE activation by 50%). Forty-eight individual compounds from the 12 positive sub-pools (inhibition rate > 50%) were then tested for their effects on SeV-induced ISRE activation by reporter assays (middle dot plots, the dashed line indicates inhibition of SeV-induced ISRE activation by >98%). The arrows indicate the workflow. The candidate compound physalin F was further tested for its dose-dependent effects on SeV-induced ISRE activation by reporter assay. The IC 50 value was calculated from the dose–response curve. IC 50 = 1.0 μM, (95% CI: 0.8 to 1.1 μM). ( B ) Effects of physalin F on transcription of downstream genes induced by SeV in HEK293T and THP1 cells. HEK293T or THP 1 cells (1 × 10 6 ) were treated with physalin F (0, 2.5, 5 μM) for 0.5 h and then infected with SeV for the indicated times before RT-qPCR analysis of mRNA levels of the indicated effector genes. GAPDH mRNA level was used as the internal control. ( C ) Effects of physalin F treatment on SeV-induced phosphorylation of TBK1, IRF3, STAT1, and STAT2. HEK293T or THP1 cells (1 × 10 6 ) were treated with the indicated concentrations of physalin F for 0.5 h and then infected with SeV for the indicated times. Immunoblotting analysis was performed with the indicated antibodies. The relative band intensities, which are normalized to the corresponding β-actin bands, are quantitated by densitometry analysis using ImageJ (1.53c) software and shown under the blots. Original Western blot images can be found in . ( D ) Effects of physalin F on IFN-γ induced transcription of the IRF1 gene in HEK293T cells. HEK293T cells (1 × 10 6 ) were treated with physalin F (0, 5 μM) for 0.5 h and then with IFN-γ for 6 h before RT-qPCR analysis of mRNA levels of the indicated antiviral genes. GAPDH mRNA level was used as the internal control. ( E ) Effects of physalin F on IFN-γ induced phosphorylation of STAT1. HEK293T (1 × 10 6 ) were treated with physalin F (0, 2.5, 5 μM) for 0.5 h and then with IFN-γ for 6 h. Immunoblotting analysis was performed with the indicated antibodies. Original Western blot images can be found in . Data shown in ( A ) (dose experiment), ( B , D ) are mean ± SD; n = 3 technical replicates. ns, not significant, * p < 0.05; ** p < 0.01. Experiments in ( B – E ) were repeated at least two times with similar results.

Article Snippet: Physalin F (TargetMol, Wellesley Hills, MA, USA, T8716); Dual-Specific Luciferase Assay Kit (Promega, Madison, WI, USA, E2490); puromycin (Thermo Fisher Scientific, Waltham, MA, USA); M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA, 28025-013); SYBR (Bio-Rad laboratories, Hercules, CA, USA); RiboLock RNase inhibitor, pyrophosphatase (Thermo Fisher Scientific, Waltham, MA, USA); protease inhibitor cocktail (Roche, Basel, Switzerland); polybrene (Millipore, Burlington, MA, USA); ClarityTM Western ECL Substrate (Bio-Rad, Hercules, CA, USA); ELISA kits for murine IFN-β (PBL Assay Science, Piscataway, NJ, USA, 42400), murine IL-6 (BioLegend, San Diego, CA, USA, 431304); mouse antibodies against HA (OriGene, Rockville, MD, USA, TA180128) and FLAG (Sigma-Aldrich, Burlington, MA, USA, F3165); mouse antibodies against β-Tubulin (ABclonal, Woburn, MA, USA, A12289); rabbit antibodies against FLAG (14793), Rig-I (3743), β-actin (5125), p-IRF3 S396 (4947), STAT1 (14994), p-STAT1 Y701 (9167) STAT2 (72604), and p-STAT2 Y690 (88410) (Cell Signaling Technology, Danvers, MA, USA); rabbit antibodies against TBK1 (ab40676), p-TBK1 S172 (ab109272), and p-IRF3 S386 (ab76493) (Abcam, Cambridge, UK); rabbit antibodies against AFG3L2 (14631-1-AP), TOM40 (18409-1-AP), TOM70 (14528-1-AP) (Proteintech, Rosemont, IL, USA), VISA (Bethyl Laboratories, Montgomery, TX, USA, A300-782A), and GFP (GeneTex, Irvine, CA, USA, GTX113617); and HRP-conjugated anti-FLAG monoclonal antibody (Sigma-Aldrich, A8592) were purchased from the indicated companies.

Techniques: Stable Transfection, Transduction, Luciferase, Infection, Inhibition, Activation Assay, Reporter Assay, Quantitative RT-PCR, Control, Phospho-proteomics, Western Blot, Software