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. 2022 Jun 2;14(3):587–607. doi: 10.1016/j.jcmgh.2022.05.012

Figure 1.

Figure 1

ALF-induced NETosis in the liver is promoted by miR-223 deficiency. (A) The formation of NETs was characterized and identified by immunofluorescence of H3Cit (red), MPO, and DAPI (blue) in liver sections from 10-week-old, male C57 BL/6J mice treated with either an intraperitoneal injection of D-GalN (0.75 mg g-1 body weight) and LPS (2.5 μg g-1 body weight) or an equal volume of PBS as vehicle. Scale bar: 5 μm. (B) Immunoblotting analysis of H3Cit in liver lysate from ALF mice or PBS-treated controls and (C) densitometric analysis of H3Cit protein abundance relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control. (D) The abundance of miR-223 in the liver of C57 BL/6J mice after treatment with either D-GalN/LPS or PBS was determined by quantitative real-time PCR. (E) NETs were detected by immunofluorescence of H3Cit, MPO, and DAPI in liver section from 10-week-old, male miR-223 KO mice and WT control mice treated with intraperitoneal injection of D-GalN (0.75 mg g-1 body weight) and LPS (2.5 μg g-1 body weight) or an equal volume of PBS as vehicle (Scale bar: 5 μm) and (F) semiquantified by the measurement of H3Cit-positive area per field. (G) Immunofluorescence of H3Cit (red), MPO, and DAPI (blue) in primary neutrophils isolated from miR-223 KO mice and WT controls with ex vivo stimulation by LPS (200 μg mL-1) to identify the formation of NETs (Scale bar: 10 μm) and (H) semiquantification under 40× microscopic fields. Ex vivo data were analyzed according to 3 independent experiments. Data in animal studies are expressed as means ± SEM. n = 5–6. ∗P < .05, ∗∗P < .01.