Pharmacologic inhibition of HIF-1α activity decreases NET formation by LPS-stimulated human PMNs. (A) HIF-1α protein expression and NET formation were simultaneously assessed with immunocytochemistry and confocal microscopy (magnification, ×60) in LPS-stimulated human PMNs (100 ng/mL), conditions that induced HIF-1α protein (Figure 2D-E). HIF-1α protein expression is shown (green fluorescence). In the merged images (bottom), extracellular and intracellular DNA are detected with TOPRO-3 (grayscale), with the yellow arrow highlighting an area of robust HIF-1α expression and NET formation. (B) NET formation was assessed by live cell imaging with confocal microscopy (magnification, ×20). Extracellular and intracellular DNA were detected with a combination of cell permeable (nuclear DNA; green) and cell impermeable (extracellular DNA; grayscale) DNA dyes. Yellow arrows highlight areas of NET formation. Human PMNs were stimulated with LPS (100 ng/mL) for 1 hour with or without pretreatment with 2-ME2 (2μM). Vinblastine (10μM) and paclitaxol (10μM), 2 inhibitors of microtubule function, served as controls for 2-ME2 pretreatment and its reported antimicrotubule activity. These images are representative of randomly selected visual fields of assays performed with PMNs from 5 different adult donors. (C) The columns represent mean ± SEM supernatant histone H3 fluorescent intensity from human PMNs isolated from 7 different healthy adult donors indicated in the graph below. The 2-sample Wilcoxon rank sum test was used. *Statistical significance with P < .05 in comparison with the LPS, vinblastine, and taxol samples.