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. 2021 Sep 8;12:702677. doi: 10.3389/fphar.2021.702677

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Copyright © 2021 Totani, Amore, Piccoli, Dell’Elba, Di Santo, Plebani, Pecce, Martelli, Rossi, Ranucci, De Fino, Moretti, Bragonzi, Romano and Evangelista.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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C57BL/6 male mice were treated as described in Figure 6 and killed after 28 h or 5 days of infection. The BALF was collected for measurement of free DNA in supernatants and citrullinated histone H3 in supernatants and in cell lysates. (A) shows box plots of free-DNA values at the time of killing (28 h and 5 days after infection) of vehicle- (n = 9 per group) or roflumilast-treated mice (n = 9 killed at 28 h and n = 8 killed at 5 days after infection). *p < 0.05 (ANOVA, Dunnett’s test) vs vehicle-treated mice. The presence of citrullinated histone H3 was analyzed by Western blotting in BALF supernatants (B) as well as in lysates of BALF cells from mice killed at 28 h and 5 days after infection (C). Pools of BALF supernatants and of cell lysates from all animals of each group were subjected to Western blotting using a monoclonal antibody which recognizes mouse with citrullinated histone H3.