Histones in the form of a nucleosome complex are not cytotoxic unless released by DNA digestion. (A) HEK293 cells were incubated overnight with histones (100 µg/mL) or nucleosomes (100 µg/mL), and lactate dehydrogenase (LDH) release was determined as a readout for cytotoxicity. Cytotoxicity was expressed as a percentage of cytotoxicity induced by 0.1% saponin. Nucleosomes or histones were preincubated with buffer and 20 nM of rFSAP, or 5000 U/mL of benzonase nuclease or a combination of both for 30 minutes at 37°C. (B-C) Nucleosomes that had been preincubated with benzonase nuclease, rFSAP, or a combination of both for the experiment in panel A were separated on agarose gel and DNA visualized with dsRED (B) or on SDS-PAGE with instant blue staining for protein (C). The gels shown are representative of 3 independent experiments. Arrows indicate the different histone subtypes and benzonase. *Indicates a 13-kDa cleavage product. (D) HEK293 cells were incubated overnight with histones (100 µg/mL) that had been preincubated with 0 to 100 µg/mL of dNTPs, ssDNA, or dsDNA for 30 minutes at 37°C. LDH release was determined as a readout for cytotoxicity. The cytotoxicity induced with 100 µg/mL of histones was defined as 100%. (E) Histones (10 µg/mL) were incubated with ssDNA, dsDNA (10 µg/mL), or buffer before incubation with sepharose-coupled anti-DNA. Histone H3 was visualized in the immunoprecipitated samples by immunoblotting. Data are expressed as mean ± standard error of the mean from 3 independent experiments, each performed in triplicate. **P < .01, ***P < .001, ****P < .0001 (calculated using a Bonferroni corrected 1-way analysis of variance). IP, immunoprecipitation.