Fig. 7. cGAMP signaling induces G1 arrest and HDR suppression.

a The distribution of WT THP1 cells in the G1, S, and G2 cell cycle phases (BrdU-FITC positivity) 24 h after stimulation with vehicle or cGAMP (n = 5 independent experiments; data presented are mean ± s.d.; two-tailed unpaired t test; *p < 0.05 indicates significance compared to respective groups; ns indicates not significant). Bromodeoxyuridine (BrdU) was added to label cells for 1 h before harvesting. Fixed cells were stained with FITC-conjugated anti-BrdU antibody and 7-AAD for total DNA content. The percentage of cells in each cell cycle phrase is shown; 20,000 cells were counted for FACS analysis. b Representative cell cycle dot plots of WT THP1 cells stimulated with vehicle or cGAMP. c Cell proliferation in human RPE cells and U2OS-STING cells after vehicle or cGAMP treatment (18 h) is measured by EdU incorporation. Cells incubated with EdU for 1 h prior to harvesting were stained for EdU incorporation using a Click-iT EdU assay. The percentages of cells with incorporated EdU as visualized by confocal microscopy are indicated in the graph. Each data point represents the percentage of cells in one image field (n = 5 fields with over 100 cells collectively per condition for hRPE cells; n = 10 fields with over 200 cells collectively per condition for U2OS-STING cells; data presented are mean ± s.d.; two-tailed unpaired t test; *p < 0.05 indicates significance compared to respective groups; ns indicates not significant). d Cell cycle analysis (propidium iodide stain) of WT and STING^−/− THP1 cells, 24 h after stimulation with cGAMP or vehicle (n = 4 independent experiments; data presented are mean ± s.d.; two-tailed unpaired t test; *p < 0.05 indicates significance compared to respective groups; ns indicates not significant). e Schematic of the experimental design utilizing a Traffic Light Reporter (TrLR) system in HEK293 cells employed to monitor DSB repair by non-homologous end joining (NHEJ) and HDR. HEK293 cells with stably integrated TrLR (HEK293-TrLR) were mock stimulated or stimulated via cGAMP transfection. Six hours post cGAMP transfection, DSBs were induced via enforced expression of the endonuclease I-SceI with or without GFP donor repair template. Seventy-two hours later, cells were trypsinized and analyzed by flow cytometry for mCherry+ or GFP+ fluorescence, indicative of NHEJ or HDR at the reporter locus, respectively. f Flow cytometric analysis of HEK293-TrLR cells transfected with vehicle/cGAMP expressing I-SceI only or I-SceI with donor. Representative graphs from n = 3 independent experiments are presented. g Quantification of data from f is presented (n = 3 Vehicle+I-SceI, n = 4 cGAMP+I-SceI, n = 5 Vehicle+I-SceI+Donor, and n = 7 cGAMP+I-SceI+Donor, Samples are from independent experiments; data presented are mean ± s.e.m.; two-tailed unpaired t test; *p < 0.05 indicates significance compared to respective groups; ns indicates not significant).