Entry of TREX1 after rupture does not cause elevation of DNA damage. (A) Overexpressed nuclease GFP-TREX1 localizes to site of nuclear rupture after constricted migration (arrow), but γH2AX foci are not enriched. A representative z-stack image confirms that TREX1 is a transmembrane protein. Bar graphs (left): Both WT and inactive (D18N) GFP-TREX1 show the same excess DNA damage after constricted migration. Functionality of WT TREX1 was confirmed by an increase in RPA2 foci on mitotic bridges of DNA (right; >150 cells per condition, n = 3 experiments; *, P < 0.05). Representative images: Overexpression of WT TREX1 (GFP-TREX1-WT) causes formation of RPA2 foci on DNA bridges (yellow arrow) after mitosis, confirming nuclease activity. With overexpression of inactive mutant TREX1 (GFP-TREX1-D18N), fewer cells exhibit such RPA2 foci. n.s., not significant. Scale bar: 10 μm. (B) Suppression of 4N is the same for WT and mutant GFP-TREX1 after constricted migration (>150 cells per condition, n = 3 experiments; *, P < 0.05).