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. 2020 Jun 1;11:2739. doi: 10.1038/s41467-020-16602-0

Fig. 6. SYNB1891 activity in human antigen-presenting cells.

Fig. 6

a–d THP-1 immortalized human monocyte cells containing an interferon-regulatory factor (IRF)-luciferase reporter and an NF-kB responsive colorimetric reporter were used. a THP-1 cells containing the endogenous STING allele (STING+/+) or lacking STING gene (STING−/−) were treated with pre-induced SYNB1891 at different ratios or media alone overnight. Cells were analyzed for the activation of the IRF reporter via luminescence with relative luminescence units (RLU). b Wildtype THP-1 cells (cGAS+/+) and those lacking the CGAS gene (cGAS−/−) were treated with pre-induced SYNB1891 or Control EcN at different ratios, or media alone overnight. Cells were analyzed for the activation of the IRF reporter and fold IRF induction in treated cells relative to untreated media alone control is shown. c, STING+/+ and STING−/− THP-1 cells were treated as described in (a) with pre-induced SYNB1891. Cells were analyzed for NFκB activity via colorimetric assay at OD655. a–c n = 2 biological replicates per group per APC: Bacterial ratio. d THP-1 cells containing an IRF-luciferase reporter and either the endogenous HAQ TMEM173 (STING) allele, knock-ins of the H232 or R232 alleles or knockout of the TMEM173 gene were treated with pre-induced SYNB1891 (MOI: 100) or media alone overnight. Cells were analyzed for the activation of the IRF reporter as described in (b) (*P = 0.0118, ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons tests, n = 4 biological replicates per group). e, f Monocyte-derived primary human DCs were treated with Control EcN (MOI: 25), pre-induced SYN1891 (MOI: 25), LPS (100 ng/mL) or smSTING agonist (5 μg/mL) for 4 h. In indicated groups cells were pre-treated for 1 h with Cytochalasin D (10 μM). Human DCs incubated in media alone served as negative control. Cells were analyzed for the upregulation of IFNB1 (e) and IL6 (f) mRNA (****P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons tests, n = 3 biological replicates per group). a–f Data are representative of two independent experiments. d–f Data with mean and s.d. shown. Each circle represents an independent experimental replicate.