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. 2020 Jul 8;11:1430. doi: 10.3389/fimmu.2020.01430

Figure 4.

Figure 4

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.