Fig. 4.

Ang II promotes NETosis and NETs secretion induced by PMA. a, b Apoptosis and autophagy analysis of human PMNs treated with PMA, Ang II of different concentrations (1 μM, 10 μM and 100 μM) or PMA+ Ang II. Analysis of p-Akt and LC3B II/I ratio (NETosis-related signaling) of PMNs treated with PMA, Ang II of different concentrations (1 μM, 10 μM and 100 μM) or PMA+ Ang II of different concentrations (1 μM, 10 μM and 100 μM) through WB (n = 3). c In vitro Ang II further upregulated PMA-induced ROS production of PMNs (n = 9). Suspended neutrophils were incubated with PMA, Ang II or PMA+ Ang II in 1.5 ml Eppendorf tubes gently shaking, and added into 96-well plate for subsequent determination of ROS with DCHF-DA in fluorescence microplate reader. d, e Analysis of concentration-dependent effect of Ang II on NETs secretion induced by PMA determined by Celigo (n = 5). Rat neutrophils in 96-well plate treated with PMA, Ang II or PMA+ Ang II of different concentrations (0.1 μM, 0.5 μM, 1 μM, 10 μM and 100 μM) were stained for all DNA (blue) and cfDNA (green). Scale bar: 150 μm. Hoechst single positive cell was defined as the living, while Hoechst and SYTOX Green double positive cell with linear DNA structure formation was defined as cells undergoing NETosis. f, g Analysis of Time-dependent effect of 10 μM Ang II on NETs secretion induced by PMA determined by Celigo (n = 4). Scale bar: 1 mm. h, i Analysis of cit-H3 in neutrophils in the presence or absence of PMA, Ang II and PMA+ Ang II through WB (n = 3). Cit-H3, citrullinated histone 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns (not significant). Data were presented as mean ± SD