Bapta am

To study the underlying signalling NET pathway, several chemical inhibitors were used. Before stimulation, neutrophils were pre-treated for 30 min at 37 °C in RPMI 10% with DMSO (vehicle control), 1 µM Wortmannin (PI3K inhibitor; Sigma-Aldrich), 1 µM R406 (SYK inhibitor; Selleckchem), 10 µM SB203580 (p38 MAPK inhibitor; Selleckchem), 10 µM SB202190 (p38 MAPK inhibitor; Santa Cruz Biotechnology) 10 µM MLN0128 (pan-mTORC inhibitor; Selleckchem), 10 µM BAPTA-AM (chelator of Ca²⁺;Focus Biomolecules), 1 µM DPI (NADPH oxidase inhibitor; Sigma-Aldrich), 1 µM Rapamycin (mTORC1 inhibitor; Selleckchem), 1 µM Celastrol (NF-κB inhibitor; InvivoGen), 0.1 µM Bay-11-7082 (IκB phosphorylation inhibitor; InvivoGen), 0.1 µM GW5074 (cRaf1 kinase inhibitor; Santa Cruz Biotechnology), 0.1 µM U-73122 hydrate (PLC inhibitor; Sigma-Aldrich), 1 nM Bafilomycin A (vacuolar-type H+ -ATPase inhibitor; Sigma-Aldrich), 0.1 µM JNKi II 420128 (JNK inhibitor; Merck Millipore) or 0.1 µM Mytoquinone (mitochondrial ROS inhibitor; BIOTREND).

The MG-63 osteosarcoma cell line was provided by RIKEN BioResource Center through the National BioResource Project of the Ministry of Education, Culture, Sport, Science and Technology of Japan (Tsukuba, Ibaraki, Japan). Anti-AKT, anti-pS473 AKT, anti-p38, anti-pT180/pY182 p38, and anti-pT202/pY204 ERK1/2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-ERK2 antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The following pharmacological inhibitors were used: BAPTA-AM (membrane-permeable calcium chelator) from Focus Biomolecules (Plymouth Meeting, PA, USA), LY294002 (PI3-kinase inhibitor) from Cayman Chemical (Ann Arbor, MI, USA), dabrafenib (RAF-1 and B-RAF inhibitor) from LKT Laboratories (Saint Paul, MN, USA), and Y-27632 (Rho kinase inhibitor) from Nacalai Tesque (Kyoto, Japan). Porcine type I collagen (Cellmatrix Type I-C) was purchased from Nitta Gelatin (Osaka, Japan). Other reagents were purchased from Nacalai Tesque unless specified otherwise.