Figure 2.

Treatment of neutrophils with AEBSF inhibits autophagy induction. (A) Immunoblot analysis of LC3B in lysates of human neutrophils pre-treated or not with AEBSF (0.35 mM; t= -30 min), then stimulated or not with LPS (t= 0); treated or not with the autophagy flux blocker Baf A1 (t= 1 h), and with ATP (t= 2 h), assessed at 2 h and 15 min post-LPS stimulation. Immunoblot is representative of 2 independent experiments. Myeloperoxidase (MPO) was employed as loading control. (B) Median fluorescence intensity (MFI) levels of the autophagy receptor p62/SQSTM1 evaluated by immunostaining and flow cytometry at 2 h and 15 min post-LPS stimulation. Data represent the mean ± SEM of experiments performed with 4 donors (left) and representative histograms of one of these experiments (right). *p < 0.05. One-way ANOVA followed by Holm-Sidak’s multiple comparisons test. (C) Representative CLSM images of neutrophils pre-treated or not with AEBSF and stimulated or not with LPS+ATP for 4 h. Cells were stained with a specific antibody anti-LC3B and the corresponding secondary antibody and DNA was stained with ToPro-3. Images are representative of experiments with 3 different donors. (D) Quantification of the images of experiments performed with 3 donors as shown in (C) Data represent the fluorescence corresponding to cytosolic LC3B expressed in arbitrary units of fluorescence (AUF). *p<0.05. Kruskal-Wallis test.