Fig. 1: cGAS prevents the formation of micronuclei.

a Generalized experimental scheme utilized to identify the role of the cGAS–STING pathway in the induction of chromosomal instability. b Thirty hours after transfection with the cGAS siRNA, nocodazole treatment (100 ng/mL) commenced for 16 h. The mitotic cell fraction was obtained by shaking the plate, and the cells were reseeded on a coverslip; the remaining cells were subjected to western blotting with the indicated antibodies (top panel). Quantification of the percentage of micronucleated cells was performed by immunocytochemistry (bottom panel) along with representative fluorescent images showing 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. c Wild-type HeLa and cGAS−/− HeLa cells were treated with 100 ng/mL nocodazole for 16 h. Mitotic cells were collected by shaking the plate, and they were then subjected to western blotting (top panel) and immunocytochemical analysis (bottom panel) to quantify micronucleated cells; representative fluorescent images of micronuclei (white arrowhead denotes micronuclei) are shown. The results are given as the mean ± SD from three independent experiments (n = 300). ***P < 0.001 as assessed by Student’s t-test. d Cells were co-transfection of sicGAS with a control vector or MYC-cGAS (non-targeting siRNA) in HeLa cells for 20 h, and then they were arrested at prometaphase by nocodazole treatment for 16 h. Western blotting with the indicated antibodies was performed to confirm cGAS knockdown and cGAS add-back (top panel). Immunocytochemistry was performed to evaluate micronucleated cells 10 h after release from nocodazole-induced mitotic arrest (bottom panel); 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. e Transfection of the MYC-cGAS plasmid into cGAS−/− HeLa cells for 12 h before the treatment with nocodazole for 16 h. Mitotic cells were collected, reseeded, and cultured for 10 h to quantify the number of cells containing micronuclei by immunocytochemistry (bottom panel); representative fluorescent images show micronuclei (white arrowhead denotes micronuclei), and their cell lysates were used for western blotting with the indicated antibodies (top 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. f U2OS cells were transfected with sicGAS for 30 h after pretreatment with nocodazole for 16 h. Mitotic cells were subjected to western blotting to determine cGAS and GAPDH (loading control) protein levels (top panel) as well as ICC to enumerate the percentage of cells with micronuclei (bottom panel); 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.