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. 2021 Jun 7;7(6):1073–1088. doi: 10.1021/acscentsci.1c00440

Figure 1.

Figure 1

SLC46A2 is a cGAMP transporter. (A) Effect of sulfasalazine (SSZ) and methotrexate (MTX) on extracellular cGAMP signaling in CD14+ monocytes. Cells were pretreated with 1 mM SSZ or 500 μM MTX for 15 min and then treated with 50 μM cGAMP for 2 h. n = 7 individual donors. (B) Diagram illustrating effects of the SLC19A1 inhibitors SSZ and MTX on extracellular cGAMP signaling in U937 cells compared to CD14+ monocytes. (C) Microarray RNA expression levels of genes annotated as plasma membrane transmembrane transporters in U937 cells compared to CD14+ monocytes. (D) Effect of SLC46A2 overexpression on extracellular cGAMP signaling. U937-tet-SLC46A2-FLAG cells were induced with 1 μg/mL doxycycline (dox) for 24 h, then treated with 50 μM cGAMP for 2 h. n = 9 biological replicates. (E) Schematic illustrating how cGAMP electroporation bypasses cGAMP transporters. (F) Effect of SLC46A2 overexpression on intracellular cGAMP signaling. U937-tet-SLC46A2-FLAG cells were induced with 1 μg/mL dox for 24 h then electroporated with 100 nM cGAMP for 2 h. n = 2 biological replicates. (G) Effect of SSZ on SLC46A2 mediated cGAMP signaling. U937-tet-SLC46A2-FLAG cells were induced with 1 μg/mL dox for 24 h, then pretreated with 1 mM SSZ for 15 min before treatment with 50 μM cGAMP for 2 h. n = 4 biological replicates. (H–J) U937-tet-SLC46A2FLAG cells were induced with 1 μg/mL dox for 24 h before treatment with either 15 μM 2′3′-cGSASMP (H), 15 μM 2′3′-CDAS (I), or 200 μM 3′3′-cGAMP (J) for 2 h. n = 2–3 biological replicates. For parts A–J, the pIRF3 signal was normalized to the tubulin signal, and data are shown as mean ± SD.