Journal: Frontiers in Immunology
Article Title: Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner
Figure Lengend Snippet: Innate Activation by M04 requires STING but not MAVS, TRIF, or cytosolic DNA PRRs. (A) Reporter assay illustrating IFN-dependent LUC induction in THF-ISRE-ΔMAVS/TRIF following overnight treatment with 1% DMSO, transfected cGAMP (10 μg/mL), or 75 μM M04. Data presented are mean ± SD relative luminescence units (RLU) using signal from DMSO-treated cells as the basis ( n = 4 treatments); (B) Immunoblot showing phosphorylation status of IRF3 Ser386, total IRF3, and GAPDH in THF-ISRE-ΔMAVS/TRIF following 8 h treatment with 1% DMSO, 75 μM M04, 1,000 HAU/mL SeV or 25 μM ABZI as indicated; (C) Reporter assay illustrating IFN-dependent LUC induction in THF-ISRE-ΔSTING following overnight treatment with 1% DMSO, 1,000 U/mL IFNβ, 1,000 HAU/mL SeV, or 75 μM M04. Data presented are mean RLU ± SD as described above; Student's T -test was used to compare RLU *** p < 0.001; (D) Immunoblot showing phosphorylation status of IRF3 Ser386, total IRF3 in THF-ISRE-ΔSTING following 4 h treatment with 1% DMSO, 50 μM M04, 1,000 HAU/mL SeV or 25 μM ABZI as indicated; (E) Secretion of bioactive type I IFN from parental THF as well as THF-ISRE-ΔMAVS/TRIF and THF-ISRE-ΔSTING treated in triplicate overnight with 1% DMSO, 1,000 HAU/mL SeV, transfected cGAMP (10 μg/mL), or 75 μM M04. Data are expressed as mean concentrations ± SD for IFNβ equivalent units. Statistical significance between treated and untreated cells of similar genetic background was calculated using Student's T -test. **** p < 0.0001; (F) Reporter assay from WT parental THF-ISRE cells as well as from cells from which indicated dsDNA-specific PRRs were deleted. Values presented are mean fold changes ± SD for duplicates relative to the value for DMSO-treated cells.
Article Snippet: Sources and concentrations of antibodies used against the following antigens are indicated in parentheses: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SC-51906; Santa Cruz) (1:10,000), IRF3 (4302; Cell Signaling), human phospho-IRF3 (76493; Abcam), mouse IRF3 (SC-9082; Santa Cruz), mouse phospho-S379 IRF3 (79945S; Cell Signaling), mouse phospho-S396 IRF3 (29047S; Cell Signaling), STING (13647S; Cell Signaling), phospho-S366 STING (19781S; Cell Signaling), TBK1 (3504S; Cell Signaling), phospho-TBK1 (5483S; Cell Signaling), NF-kB P65 (SC372; Santa Cruz), NF-kB P50 (3035; Cell Signaling), GM-130 (610823; BD Biosciences).
Techniques: Activation Assay, Reporter Assay, Transfection, Western Blot