Fig. 1. Nuclear localization and DNA binding activity of cGAS are required to suppress cell proliferation, independently of STING and cGAMP.

(A and B) Western blot (WB) of cGAS in WT and cGAS^−/− BJ and MEF cells. Thirty-thousand cells were seeded into each well of a six-well plate. The number of BJ WT/cGAS^−/− (A) and MEF WT/cGAS^−/− (B) cells was counted on the indicated day to determine the average population doubling time. (C) WB of STING in BJ and U2OS WT cells and cGAS in WT, cGAS^−/−, and green fluorescent protein (GFP)–cGAS stably overexpressed WT U2OS cells (cGAS-OE). Proliferation rates of WT, cGAS^−/−, and cGAS-OE U2OS cells were determined as described in (A) and (B). (D and E) Schematic structure of cGAS truncated proteins. WB of flag-tagged cGAS deletions and mutants in U2OS cGAS^−/− cells is shown. (F) Thirty-thousand cells were seeded into each well of a six-well plate. The number of U2OS cGAS^−/− cells transfected with flag-tagged deletion and flag-vector plasmids was counted every day until reaching high density. (G) U2OS cGAS^−/− cells were transfected with flag-tagged cGAS point mutant plasmids and tested by immunostaining (scale bar, 10 μm). (H) Quantification of percentage of cells with nuclear cGAS (n = 50). Three independent experiments were done. (I) Thirty-thousand cells were seeded into each well of a six-well plate. The number of U2OS cGAS^−/− cells transfected with flag-tagged full length (FL), Y215E, K347E, K394E, and flag-vector plasmids was counted every day until reaching high density. Note that the same vector control is used in (F) and (I). ***P < 0.001, Mann-Whitney test.