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. 2020 Jan 28;11(1):e03187-19. doi: 10.1128/mBio.03187-19

FIG 1.

FIG 1

cGAS-independent, STING-dependent transactivation of macrophages by cGAMP-producing cells. (A) BMDMs (WT, Sting−/−, or cGas−/−) were cocultured for 18 h with Sting−/− MEFs that were previously transfected with 2 μg/ml of immunostimulatory DNA (ISD) for 2 h (and extensively washed to remove any residual ISD, as previously reported [21]). The mock condition was Lipofectamine only. Murine IP-10 (IP-10) (right panel) or type I IFN activity (left panel) levels were measured from the supernatant of the different coculture conditions. (B and C) The functionality of cGas-Sting signaling in BMDMs (B) or MEFs (C) was validated by IP-10 enzyme-linked immunosorbent assay (ELISA) following transfection of 2 μg/ml ISD for 18 h. (A to C) Data are averaged from a minimum of two independent experiments conducted in biological triplicate (± the standard errors of the mean [SEM] and unpaired t test results compared to mock conditions are shown). (D) IP-10 levels were measured from the supernatants of HEK or HEK-cGAS cells cocultured for 18 h with PMA-treated THP-1 monocytes (matched WT and STING−/− [D, left panel] or cGAS−/− [D, right panel]). The data shown are averaged from three independent experiments conducted in biological triplicate (± the SEM). (E) Primary human monocytes (mono) from two donors or THP-1 WT cells were cocultured for 18 h with HEK or HEK-cGAS cells and IFN-β levels measured from the supernatant. The data shown are averaged from two independent experiments conducted in biological duplicate and representative of three blood donors (primary monocytes), or are averaged from three independent experiments conducted in biological triplicate (THP-1) (± the SEM and unpaired t tests comparing HEK to HEK-cGAS coculture). (F and G) PMA-treated WT THP-1 cells (F) or cGAS−/− cells (G) constitutively expressing the fluorescent protein citrine (an EGFP variant) were cocultured with HEK or HEK-cGAS for 9 h before being sorted by flow cytometry based on citrine expression. Pure populations of WT THP-1-citrine cells (F) or cGAS−/−-citrine cells (G) were harvested to analyze expression of a panel of ISGs by RT-qPCR. cGAS−/− THP-1-citrine cells were also harvested to determine the level of intracellular cGAMP by specific ELISA (G, right panel). #, The limit of detection by the cGAMP ELISA. (F and G) Data shown are averaged from two independent experiments conducted in biological duplicate. (A to G) Each point represents the mean data for each independent experiment; the column represents the mean of the experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001, ns, nonsignificant. Detailed materials and methods are provided in Text S1.