Fig. 3: cGAS activity-dependent downregulation of the micronuclei phenotype.

a cGAS−/− HeLa cells were transfected with the indicated plasmids for 6 h and then were treated with nocodazole for 16 h. Then, mitotic cells were collected by shaking the plate, and they were subjected to western blotting (left panel) with the indicated antibodies. Further, ICC (right panel) was performed to determine the percentage of cells showing micronuclei 10 h after release from nocodazole, and representative fluorescent images show micronuclei (white arrowhead denotes micronuclei). The results are given as the mean ± SD from three independent experiments (n = 300). ***P < 0.001 as assessed by Student’s t-test. b cGAS−/− HeLa cells were transfected with or without cGAMP using Lipofectamine® 2000 transfection reagent and were then incubated for 6 h. Then, the cells were arrested at the M phase via nocodazole treatment for 16 h. A portion of the mitotic cells were seeded on a coverslip to enumerate the cells showing a micronuclei phenotype 10 h after release from arrest, as assessed by ICC (right panel) along with representative fluorescent images showing micronuclei (white arrowhead denotes micronuclei). The remaining portion of cells was subjected to western blotting with the indicated antibodies (left panel). The results are given as the mean ± SD from three independent experiments (n = 300). ***P < 0.001 as assessed by Student’s t-test. c HeLa cells transfected with sicGAS for 20 h were transfected with or without cGAMP for 6 h and then subjected to nocodazole treatment for 16 h. Ten hours after release from nocodazole treatment, western blotting (left panel) with the indicated antibodies and ICC (right panel) was performed to quantify cells displaying a micronuclei phenotype. The results are given as the mean ± SD from three independent experiments (n = 300). ***P < 0.001 as assessed by Student’s t-test.