Extended Data Fig. 3: Unlike PMA-induced suicidal NET release LTB4-induced DNA secretion is nonlytic and is PAD4- and NOX2-independent.
From: Neutrophils secrete exosome-associated DNA to resolve sterile acute inflammation
a-b. Time-lapse of PMNs stained with Mitotracker red CMXros (red, mitochondria), Hoechst (blue, nuclei), and SYTOXgreen (green, extracellular DNA) (a) migrating towards LTB4 or (b) treated with 100 nM PMA. Insets in panel b show cell outline (red), nuclei (blue), and NETs (green) generated by CellProfiler. Scale is 5 μm. Images are representative of three independent experiments. See associated Supplementary Movies 3 and 5. c. Before-after aligned dot plot showing Mitotracker Red intensity in migrating PMNs before, during, and after DNA secretion within 1 hr. Total 30 data points (red circles) pooled from three independent experiments are presented as mean ± s.e.m. (black lines), with multiplicity-adjusted P values from ordinary one-way ANOVA. d. Scatter dot plot showing percentage of DNA-secreting PMNs chemotaxis towards LTB4 with or without MitoTEMPO (10 μM). Data points (red dots) from three independent experiments are presented as mean ± s.e.m. (black lines), with P value from two-tailed ratio paired t-test. e. Airyscan microscopy image of Mitotracker Red CMXros (magenta) stained PMNs chemotaxing towards LTB4, fixed and immunostained for FLAP (yellow) and Hoechst 33342 (cyan). Orange dashed line outlines cell and inset is zoomed as individual channels (invert grayscale). The 3D-volume is shown below. Images are representative of three independent experiments and scale is 5 μm. f. Graph showing nuclei area extent over time in PMNs treated with PMA or migrating towards LTB4. Dots represent mean nuclear area extent per 40,000 μm2. Blue/red dots indicate percentage of PMNs without/with SYTOXgreen staining. Thick black line represents non-linear regression. Data is representative of three independent experiments. g. Graph showing the percentage of PMNs lysed over time. Data points from three independent experiments are plotted as mean (red/blue dots) ± s.e.m. (black lines). h. Graph showing the percentage of maximum lactate dehydrogenase (LDH) activity in supernatants of PMNs treated with either DMSO, PMA (20 nM), or LTB4 (100 nM) for 2 hrs. Data points from three independent experiments (similarly coloured circles) are presented as mean ± s.e.m. (black lines), with multiplicity-adjusted P values from RM one-way ANOVA. i-j. Scatter dot plot showing percentage of DNA-secreting PMNs migrating towards LTB4 in a 40,000 μm2 observation window for 1 hr either (i) with or without PAD4 and NOX2 inhibitors, GSK484 (2 µM) and GSK2795039 (10 µM), respectively, or (j) cell death pathway inhibitors namely, Z-DEVD-FMK (apoptosis, 10 μM), ferrostatin-1 (ferroptosis, 5 μM), and GSK872 (necroptosis, 10 μM). Data points from three (or more) independent experiments (similarly coloured circles) are presented as mean ± s.e.m., with multiplicity-adjusted P values from RM one-way ANOVA (i) and mixed-effect analysis (j). See associated Supplementary Movie 6 and 7. Source numerical data is available in the source data file.
