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. 2022 May 24;11:e72103. doi: 10.7554/eLife.72103

Figure 2. IFI16 and AIM2 bind NETs and prevent NET degradation by DNase I.

Figure 2.

NETs were induced in neutrophils using PMA 100 nM for 3 hr, then left untreated (A) or incubated with fluorescently labeled AIM2 (pink) and IFI16 (red) at 200 nM at RT for 1 hr (B). Following ALR incubation, samples were stained with anti-MPO-FITC antibody (green) and DAPI (blue), then imaged by confocal microscopy. NETs were treated with DNase I at 20 U/mL at RT for 1 hr (C). NETs incubated with ALRs as in (B) were then treated with 20 U/mL DNase I for 1 hr (D). Scale bars = 20 µm. NETs in 96 well plates were incubated with ALRs at 200 nM (or buffer only) for 1 hr at RT, then treated with DNase I at 0, 20, and 100 U/mL for 30 min at RT. NETs were then stained with Sytox-Green 5 µM, and samples analyzed by fluorimetry (E). RFU = fluorescence units. Mean and standard deviation of 4 replicate wells are indicated. Mann-Whitney test was used to compare groups. P > 0.05 = not significant (ns). P < 0.05 = significant (*). IFI16 and the catalytic domain of cGAS were combined with FAM-labeled 72 bp VACV dsDNA for 30 min, then DNase I added at concentration of 20 U/mL at time = 0 and the fraction of bound dsDNA was monitored via the fluorescence anisotropy of dsDNA•protein complex (F).