Figure 4.

The AMPK/p38/autophagy axis regulates in vitro NET formation in response to H₂O₂ stimulation. Inhibition of phosphorylation of AMPK and p38 impairs NET formation. A) Representative fluorescent images of WT neutrophils pretreated for 30 min with 10 µM of phospho-p38 inhibitor SB203580 (p38-i) or 10 µM of phospho-AMPK inhibitor Compound C (AMPK-i) followed by stimulation with H₂O₂. NETs were visualized using Sytox Green staining. Original magnification, ×200. Bar graph shows quantification of NETs (average ± se) from 3 independent experiments. ***P < 0.001. B) Western blot analysis of autophagy activation indicated by level of LC3-II expression in WT neutrophils stimulated with 10 mM H₂O₂ in the presence or absence of p38-i and AMPK-i. Bar graph depicts densitometry analysis of protein bands showing the ratio of LC3-II over LC3-I after normalization of band intensities using that of β-actin. Data shown is the average of 3 to 4 independent experiments. C) Western blot analysis of phospho-p38 expression in H₂O₂-stimulated neutrophils in the presence or absence of AMPK-i. Bar graph depicts densitometry analysis protein band intensities normalized to β-actin. D) Western blot analysis of phospho-AMPKα1, phospho-p38, and LC3-I and -II expression in H₂O₂-stimulated neutrophils in the presence or absence of DPQ. Bar graph shows densitometry of specific band intensities normalized to β-actin. E) A model depicting signaling pathway of TRPM2-mediated NET formation in response to ROS.