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. 2021 Sep 29;12:746032. doi: 10.3389/fimmu.2021.746032

Figure 1.

Figure 1

Neutrophils resist inflammasome-dependent cell death. (A) Quantification of IL-1β in the culture supernatants of mouse bone marrow-derived neutrophils (BMNs) or macrophages (BMDMs) untreated (Unt) or primed with LPS (0.25 μg/mL, 2.5 h), followed by ATP (2.5 mM, 1 h) or nigericin (Nig, 5 μM, 1 h) treatment, or flagellin (500 μg/mL, 6 h) or poly dA:dT (1 μg/mL, 6 h) transfection (n = 3). (B) Immunoblots of mouse BMNs or BMDMs treated as in (A). Culture supernatants (Sup) or cell lysates (Lys) immunoblotted with the indicated antibodies. The asterisk indicates a nonspecific band. (C, D) Quantification of LDH in the culture supernatants of mouse BMNs or BMDMs treated as in (A) (C, n = 3) or primed with LPS (0.25 μg/mL, 2.5 h) and treated with ATP (2.5 mM, 1, 3, 6 h) (D, n = 3-5). (E) Quantification of LDH in the culture supernatant of mouse splenic neutrophils or BMDMs primed with LPS, followed by ATP treatment (0.5 or 2 h). (n = 3) (F) Quantification of PI-positive mouse BMNs or BMDMs primed with LPS, followed by ATP (2.5 mM, 1 h) or treated with staurosporine (STP, 2 μg/mL, 6 h), as determined by flow cytometric analysis after propidium iodide (PI) staining. (n = 3). (G) Immunoblots of cell lysates from mouse BMNs or BMDMs primed with LPS followed by ATP (1 or 3 h), or staurosporine (STP, 2 μg/mL, 6 h) treatment. (H) Quantification of phosphatidylserine-positive mouse BMNs or BMDMs treated with LPS (0.25 μg/mL), followed by ATP (2 h) or staurosporine (STP, 5 h), as determined by flow cytometric analysis after Annexin V-FITC staining (n = 2 or 3). (I) Quantification of LDH release into the culture supernatant of mouse BMNs or BMDMs incubated with culture medium (24 h), treated with LPS (0.25 μg/mL, 24 h) in the presence of zVAD (20 μM) or treated with LPS (0.25 μg/mL, 2.5 h) followed by ATP (2.5 mM, 3 h) (n = 3). Data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant.