Fig. 2
From: Neutrophil extracellular trap formation requires OPA1-dependent glycolytic ATP production
Absence of microtubule formation in OPA1-deficient human and mouse neutrophils. a Super-resolution microscopy. Microtubule formation and mitochondrial morphology of primary mature neutrophils from Opa1N∆ and control mice following GM-CSF priming and subsequent C5a stimulation. Microtubules were stained with anti-α-tubulin antibody (green), the nucleus with Hoechst 33342 (blue) and mitochondria with MitoTracker® Orange (red). Images were acquired by ELYRA super-resolution microscopy. Bars, 10 µm. The data are representative of three independent experiments. Quantification and additional data including microtubule formation induced by other triggers are provided in Supplementary Fig. 4a–c. b Super-resolution microscopy. Microtubule formation and mitochondrial morphology in human blood neutrophils from control individuals and an ADAO patient (son, see Fig. 1) following GM-CSF priming and subsequent C5a stimulation. Microtubules were stained with anti-α-tubulin antibody (green), the nucleus with Hoechst 33342 (blue) and mitochondria with MitoTracker® Orange (red). Single cells are shown at higher magnification in the insets. Images were acquired by ELYRA super-resolution microscopy. Bars, 10 µm. The data are representative of three independent experiments. Quantification is provided in Supplementary Fig. 5a. c Confocal microscopy. Microtubule assembly was analyzed in human control neutrophils following pre-treatment with the indicated inhibitors and subsequent combined GM-CSF/C5a stimulation. Microtubules were stained with anti-α-tubulin antibody (red), the nucleus with Hoechst 33342 (blue). Bars, 10 µm. Right: Quantification of the microtubule network formation was performed by automated analysis of microscopy images using Imaris software. Values are means ± SEM. n.s., not significant; ***p < 0.001; n = 5. d Confocal microscopy. Human blood neutrophils from control individuals pretreated with the indicated inhibitors were stimulated with GM-CSF/C5a. Extracellular DNA was stained with MitoSOX™ Red and the nucleus with Hoechst 33342 (blue). Bars, 10 μm. Right: quantification of the released dsDNA in supernatants of activated neutrophils. Values are means ± SEM. n.s., not significant; ***p < 0.001; n = 3
