Fig 1: Conditions that result in collided ribosomes induce cytosolic localization of cGAS(A) U2OS cells stably expressing GFP-cGAS were transfected with the indicated siRNA. Cells were fixed, stained with eS8 antibody or with DAPI, and imaged by confocal fluorescence microscopy. Scale bar: 10 μm.(B) As in (A), but after treatment with the indicated drug regimens.(C) As in (A) and (B), but after acute heat shock treatment. Quantification of Figures S8A–S8C.(D) Analysis of the interaction between cGAS and ribosomes using the in situ proximity ligation assay (PLA) before and after heat shock in U2OS cGAS KO cells stably expressing FLAG-hemagglutinin (HA)-tagged GAS. Cells were fixed, incubated with the indicated antibodies, and visualized according to instruction of Dulink In Situ Kit. The PLA signal was detected by confocal fluorescence microscopy. Scale bar: 10 μm.(E) Quantitative analysis of (D). Two-tailed t test, ∗∗∗∗p < 0.0001. Error bars represent SD of puncta per cell from 80 cells per condition.(F) qRT-PCR analysis of relative ISG expression in U2OS cells treated with heat shock. Error bars represent SD of three technical replicates and are representative of three biological replicates.(G) As in (F), but cells are also treated with translation inhibitor cycloheximide (CHX).ns, no significant. See also Figure S8 for quantification.