Figure 3.

Targeted multi-omics analysis of neutrophils. Neutrophils isolated from healthy donors (n = 3) were treated with PMA (25 nM) or ionomycin (4 µM). The cell supernatants were isolated and analyzed. Error bars indicate standard deviations. * indicate p-value < 0.05. (A) Proteomics. The fold changes of NETs proteins and S1008/9 proteins in treated versus untreated samples are shown. (B) Metabolomics. The fold changes of six metabolites in treated versus untreated samples are represented. (C) Eicosadomics. The abundances of selected eicosanoids as total areas normalized to global deuterated standards (TAN) are blotted. Neutrophils (n = 3) were cultured in RPMI medium supplemented with 0.01% FCS. At all treatment conditions, eicosanoids were significantly up-regulated when comparing treated samples with their respective untreated control samples. (D) Immunolocalization of spermine/spermidine in neutrophils. Comparison between neutrophils which were untreated CONTROLS or activated for one hour with PMA or IONOMYCIN. Spermine/spermidine (Spe/Spd) is depicted in white, mitochondria, through staining of mitochondrial import receptor subunit TOM20 homolog (TOMM20), in red, and endoplasmic reticulum (ER) in green. Nuclei are counterstained in blue with DAPI. Merged images are provided as fluorescence confocal images with or without transmitted light channel for visualization of cellular morphology in addition to the structures of interest. Scale bars stand for 5 µm. (E) Levels of spermidine (Spd) and spermine (Spe) in mitochondria and endoplasmic reticulum (ER). Mitochondria and ER were isolated by sucrose gradient from lysed treated and untreated neutrophils, respectively. Levels of Spd and Spe were determined in these organelles and compared between treated and untreated cells, as represented.