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. 2016 May 27;7:11752. doi: 10.1038/ncomms11752

Figure 1. The ssDNA-binding of RPA and Rad51 prevents type I IFN activation caused by leakage of nuclear ssDNA into the cytosol.

Figure 1

(a,b) The cytoplasm (a) or the nucleus (b) of HEK293T cells was microinjected with ssDNA647N (ssDNA, red) and FITC-dextran (dextran, green) as tracer. Images were taken at the indicated time points. Scale bar, 10 μM. (c) Representative image of a binuclear HEK cell. Fluorescence of ssDNA647N (ssDNA, red) microinjected into one nucleus, marked by FITC-dextran (dextran, green), is taken up by the non-injected adjacent nucleus. Scale bar, 10 μM. (d) ssDNA647N (ssDNA) microinjected into nuclei of HeLa cells with knockdown of RPA70 and Rad51 (siRPA+siRad51) leaks into the cytosol within 15 min, but remains nuclear in cells transfected with negative control siRNA (siCtrl). Co-injected FITC-dextran (green) marks the injected compartment. Scale bar, 10 μM. (e,f) Phosphorylation of IRF3 and p53 (e) as well as IFN-β induction (f) after knockdown of RPA70 and Rad51 (siRPA, siRad51) is suppressed by additional knockdown of cGAS. siCtrl, negative control siRNA. (f) Shown are the means of one experiment run in triplicates out of two independent experiments. Error bars, s.d. ***P<0.001 versus siCtrl. ###P<0.001 versus knockdown of RPA70, Rad51 or RPA70 and Rad51, respectively, by analysis of variance followed by Tukey' post hoc test.