Genetic inhibition of HIF-1α activity decreases NET formation by LPS-stimulated surrogate PMNs. (A) Differentiated HL-60 surrogate PMNs were transduced with a lentivirus designed to coexpress GFP (as a marker for transduction) and an shRNA targeting HIF-1α (HIF-1αKD) to inhibit HIF-1α expression. HL-60 surrogate PMNs transduced with GFP lentivirus encoding a scrambled shRNA served as controls (SC Control). HIF-1α protein expression was determined with the In-Cell Western technique. The results are presented as fluorescence/cell ± SEM. *P < .05. We used Student t test to compare HIF-1α protein expression in HIF-1αKD and SC Control cells. (B) We also compared LPS-stimulated HIF-1αKD and SC Control cell expression of HIF-1α protein with the use of semiquantitative immunocytochemistry. The results are presented as fold change over baseline fluorescence/cell ± SEM. *Statistical significance, P < .05, using Student t test. (C) We assessed NET formation by LPS-stimulated HIF-1αKD and SC Control surrogate PMNs with the use of live cell imaging with confocal microscopy (magnification, ×20). Extracellular DNA was detected with a cell impermeable DNA dye (extracellular DNA; grayscale). Green fluorescence represents GFP expression. Yellow arrows highlight areas of NET formation. (C) We used Western blotting to quantify extracellular histone H3 content in DNase-treated supernatants as a surrogate for NET formation by HIF-1αKD and SC Control cells. This image is representative of assays performed in surrogate PMNs isolated from 4 discrete differentiation cultures with densitometry readings from all experiments represented in the graph below. (D) Columns represent supernatant histone H3 fluorescent intensity ± SEM. The Kruskal-Wallis equality-of-populations rank test with 2-sample Wilcoxon rank sum posthoc testing was used. *Statistical significance (P < .05) in comparison of both control and LPS-stimulated samples. The results presented in this figure represent at least 4 separate experiments performed with surrogate PMNs.