Extended Data Fig. 3: SLC19A1 is critical for CDN-induced gene expression.

a, b, mRNA expression levels of SLC19A1 (a) or IRF3 (b) in THP-1 cells expressing a CRISPRi vector and a control non-targeting gRNA or gRNAs targeting IRF3 or SLC19A1 (two gRNAs each). c, THP-1 cells described in a and b were exposed to [³H]-methotrexate. After 1 h, radioactivity (in counts per minute, cpm) in lysates of cell pellets was measured. Counts were normalized to protein concentrations in the lysate. d, THP-1 cells expressing a control gRNA or SLC19A1-targeting gRNA were exposed to indicated concentrations of 2′3′-RR CDA or 2′3′-cGAMP. After 20 h, the mean fluorescence intensity (MFI) of tdTomato was quantified by flow cytometry. e, THP-1 cells expressing the indicated CRISPRi gRNAs or non-targeting gRNA (control), were stimulated with IFN-β (100 ng ml⁻¹). After 18–22 h, tdTomato expression was quantified as in Fig. 2a. f, U937 cells expressing the indicated CRISPRi gRNAs or non-targeting gRNA (control), were stimulated with 2′3′-RR CDA (1.67 μg ml⁻¹), 2′3′-cGAMP (15 μg ml⁻¹) or IFN-β (100 ng ml⁻¹). After 18–22h, tdTomato expression was quantified as in Fig. 2a. g, h, Induction of CXCL10 mRNA (g) or CCL5 mRNA (h) in control (non-targeting gRNA) THP-1 cells or THP-1 cells expressing the indicated CRISPRi gRNAs after 5 h stimulation with 5 μg ml⁻¹ 2′3′-RR CDA. i, CXCL10 protein expression in the supernatant of indicated gRNA-expressing THP-1 cells after exposure to 2 μg ml⁻¹ 2′3′-RR CDA for 20 h. j, Various cell lines expressing a control vector or an SLC19A1 expression vector were stimulated with IFN-β (100 ng ml⁻¹). After 18–22 h, tdTomato expression was quantified as in Fig. 2a. k, THP-1 cells were incubated with increasing concentrations of the non-competitive inhibitor sulfasalazine or DMSO as vehicle control, before stimulation with 2′3′-RR CDA (1.25 μg ml⁻¹), 2′3′-cGAMP (15 μg ml⁻¹) or IFN-β (100 ng ml⁻¹). After 18–22 h, tdTomato expression was quantified as in Fig. 2a. The data were normalized to the DMSO controls. In a–c and e–f, mean of n = 2 biological replicates are shown. In d, g–k, mean ± s.e.m. of n = 3 biological replicates are shown. Statistical analyses were performed to compare each cell line to the control using a one-way ANOVA followed by post hoc Dunnett’s test (d, g–i) or two-way ANOVA followed by uncorrected Fisher’s least significant difference tests (j). ****P ≤ 0.0001; n.s., not significant.

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