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. 2021 Mar 4;12:613597. doi: 10.3389/fimmu.2021.613597

Figure 2.

Figure 2

In vivo chromatin clearance is delayed in DNase1-deficient mice. (A) Chromatin is recovered in plasma from chromatin-injected mice. Four wild type (WT) mice were injected intravenously with purified mono-/di-nucleosomes. Plasma was prepared from tested mice before (0) and 30 min after injection. DNA was isolated from individual mice and analyzed by agarose gel electrophoresis. For comparison, purified nucleosomes (Nuc) used for injections were loaded. One representative of four independent experiments is depicted. The different lanes shown were extracted from the same gel without modification of brightness. bp, base pair; M, molecular weight marker (100 bp ladder). (B) Kinetics of chromatin clearance in three wild type and three DNase1-deficient [KO (knockout), namely DNase1−/−/Trap1m/m] mice. Mice were injected intravenously with purified oligo-nucleosomes and plasmas were prepared 2 and 30, 2 and 60, or 2 and 90 min post injection. One mouse was used for one time point (thus, each mouse was bled two times post injection). DNA was isolated and analyzed as in (A). For comparison, purified chromatin (chrom) used for injections was loaded. Shown is one representative experiment of three independent experiments, each with six mice (three mice per group). (C) DNA isolated from plasma in (B) was quantified by measuring mean intensities and pixels of each signal on pictures from agarose gels and normalized to the 2-min time point for each mouse; Wild type (n = 3, blue triangles) and DNase1−/−/Trap1m/m (n = 3, pink circles) mice are depicted, with one mouse per time point in each group. (D) Clearance of circulating chromatin was analyzed as in (B,C) at 60 min post chromatin injection in 26 independent mice, both in wild type (n = 13, blue triangles) and DNase1−/−/Trap1m/m (n = 13, pink circles) mice. For each mouse, the 60-min signal was normalized to the 2-min signal. Shown are data pooled from four independent experiments, with three independent chromatin preparations. Mean and SEM are depicted. *p < 0.05 (two-tailed unpaired t-test).