Necrostatin-1 Alleviates Diffuse Pulmonary Haemorrhage by Preventing the Release of NETs via Inhibiting NE/GSDMD Activation in Murine Lupus

Figure 2

Nec-1 inhibited the formation of neutrophil extracellular traps (NETs) in vitro. (a) Neutrophils were cultured with phorbol 12-myristate 13-acetate (PMA) or PMA plus Nec-1 (50 μM or 100 μM). Representative immunofluorescence images of neutrophils stained using SYTOX Green (green) and Hoechst (blue) (200x). (b) Peripheral blood neutrophils were cultured with PMA or PMA plus different concentrations of Nec-1 (50 or 100 μM). Extracellular DNA levels, which were determined via SYTOX Green staining, were detected using a microplate reader. (c) SYTOX Green-stained neutrophils stimulated with 25 nM PMA or with different concentrations of Nec-1 (50 or 100 μM) were photographed for 4 h using an automatic live-cell imaging analysis system with the GFP green channel (measurement of non-transmembrane SYTOX Green). The green fibrin-like structures indicate NETs. Videos S1–S4 show the change of untreated cells and also of cells treated with PMA (25 nM), PMA+Nec-1 (50 μM), and PMA+Nec-1 (100 μM), respectively. (d) The mean fluorescence intensity of live-cell imaging pictures at different time points is presented and analysed (0-240 min). The mean fluorescence intensity of SYTOX Green at different time points was determined using ImageJ semiquantitative statistics. Values are expressed as error of the mean (SEM) of three independent experiments ( and ).

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