Figure 5. Z-DNA binds to ZBP1, activates the cGAS-STING pathway and has stronger immunostimulatory properties than B-DNA.

A-B. Gene expression of ZBP1 after IFNα stimulation in N/TERTs (A.) and primary healthy control KCs (B.) compared to β-Actin. C. Representative image of confocal microscopy from N/TERTs preincubated with IFNα (1000U/ml) and then irradiated with UVB exposure stained for Z-DNA, ZBP1, and DAPI 3h after UVB exposure. Scale bar 5μm. D. Representative images of IFNα-treated N/TERTs stained for Z-DNA, cGAS and DAPI 3h after UVB exposure. Cellular outline was drawn based on CellTrackerRed counterstain. E. Gene expression at 6h of indicated genes from N/TERTs and STING KO N/TERTs treated with Lipofectamine2000 alone or transfected with Z-DNA or B-DNA (500ng/ml). F. Representative Western Blot (n=2) of indicated proteins from N/TERTs transfected with 50ng (low) or 500ng (high) of Z-DNA (polydGdC) or B-DNA using Lipofectamine 2000. Lysates harvested 4h after transfection. G. Quantification of the abundance of pSTING, pIRF3, and pTBK1 relative to unphosphorylated proteins in transfected KCs. H. N/TERTs were transfected as in E and gene expression was measured 6h after DNA transfection (n=3). I. Primary control KCs (n=3) were transfected and analyzed as in H. Unpaired t-test and Ordinary one–way ANOVA followed by Sidak’s multiple comparison test. Mean and SEM. *P<0.05, **P<0.01, ***P<0,001, ****P<0.0001.