Fig. 4: GAPDH inhibition inhibits the neutrophil respiratory burst and causes NOX-independent NET formation.

a, b Dose-dependent effect of HA on TNF-α + fMLF-induced ROS production in the respiratory burst as measured by lucigenin signal intensity in primary human neutrophils. A representative trace from five independent experiments is shown in (a) and quantitation of four samples/treatment from two independent experiments using 100 μM HA shown in (b). ROS reactive oxygen species. c Schematic of methylglyoxal (MGO) production from DHAP. d Methylglyoxal levels in primary human neutrophils after HA treatment. n = 4 donor samples/treatment in four independent experiments. e Methylglyoxal labeling from U¹³C-glucose tracing. n = 7 samples/treatment from five donors in five independent experiments. f, g Dose-dependent effect of methylglyoxal on TNF-α + fMLF-induced ROS production in the respiratory burst. A representative trace (f) and quantitation of independent experiments using 100 μm methylglyoxal (g) are shown. n = 6 samples/treatment from three donors in three independent experiments. h Effect of methylglyoxal on TNF-α + fMLF-induced ROS production in the respiratory burst as measured by ROS sensors using flow cytometry from n = 3–4 independent experiments. i Effect of a G6PD inhibitor (G6PDi) on PMA-induced or HA-induced NET formation assessed with a Sytox Green microplate assay. n = 6 samples/treatment from three donors in three independent experiments. j Effect of NOX inhibition by DPI on PMA-induced or HA-induced NET formation assessed with a Sytox Green microplate assay. n = 10 samples/treatment from five donors in five independent experiments. k Effect of NOX inhibition by DPI on PMA-induced or HA-induced NET formation assessed by time-lapse microscopy. Shown is one frame/condition from time-lapse movie. Time-lapse frames are shown in the Supplementary Figs. n = 3 independent experiments. Data represent mean ± st.dev. Statistical significance was assessed with two-tailed Welch’s t-test on log-transformed values (b, g), two-tailed paired t-test of log-transformed values (d), two-tailed Welch’s t-test (for m + 3 in panel e), one-way ANOVA of log-transformed values with Holm–Sidak multiple comparisons correction (h), two-tailed paired t-test (i, j). For some of the experiments in panels i and j, G6PDi and DPI treatments were tested in parallel with the same PMA or HA treatments.