(a) Endogenous cGAS was stained by immunofluorescence of U2OS cells in mitosis, showing a diffuse staining pattern without accumulation at the DAPI stained condensed chromosomes at metaphase. Two representative images shown. During anaphase/telophase cGAS staining can be seen on DNA in some cells. Overexpressed GFP-cGAS is also observed to localise more widely to mitotic DNA in U2OS cells and MEFs (data not shown). (b) Quantification of cGAS staining during mitosis, by stage. (c) Rnaseh2b-/- p53-/- MEFs stably expressing GFP-cGAS show localisation of cGAS at DNA bridges (orange arrowheads). (d) Endogenous cGAS can also be seen to localise to DNA bridges that occasionally occur in U2OS cells. cGAS also localised to micronuclei that occurred in the same cells (green arrow heads). Interphase chromatin bridges with cGAS bound in Rnaseh2b-/- p53-/- MEFs 0.08% of n=1,223 cells; U2OS cells 0.06% of n=1,632 cells. Scale bars, 10 μm.