Peg catalase

For inhibition experiments, cells were isolated, settled in RPMIcomp. supplemented with 10 mM HEPES and activated as described above. Inhibitors or ROS scavengers were added at least 20 min (in case of MitoTEMPO 1 h) before cell irradiation with 70 J/cm² of 375 nm or 214 J/cm² of 470 nm, at 37°C. For an additional control experiment, Trolox and catalase/SOD were added separately after irradiation. The cells were then incubated for an additional 3 h without an additional washing step in the presence of the inhibitors to allow for NET formation and fixed by 2% PFA. Pure medium or 100 nM PMA without irradiation were used as negative and positive controls, respectively. The following inhibitors and ROS scavengers were used in this study: GW-311616A hydrochloride (iNE, Axon Medchem) at 5 μM, 4-aminobenzoic acid hydrazide (4-ABAH, Cayman chemicals) at 100 μM, z-VAD-FMK (Promega) at 20 μM, necrostatin-1 (Nec-1, Enzo) at 50 μM, Y-27632-dihydrochloride (Abcam) at 20 μM, Cl-amidine (Merck Millipore) at 200 μM, MitoTEMPO (Sigma-Aldrich) at 5 μM, diphenyleneiodonium chloride (DPI, Sigma-Aldrich) at 1 μM, Trolox (Sigma-Aldrich) at 50 μM, PEG-catalase at 2,000 U/ml (Sigma-Aldrich), and a mixture of catalase (filtered, Worthington) and superoxide dismutase (SOD, Sigma Aldrich/Merck) at 2,000 and 50 U/ml, respectively.

Primary human aortic adventitial fibroblasts (AoAF; Lonza no. CC-7014; Lonza) were grown in Stromal Cell Growth Medium (SCGM; Lonza no. CC-3205), containing 5% FBS and used from passages 2 to 8. Fibroblasts were maintained in T-75 flasks in a humidified incubator at 37°C with 5% CO₂. Once confluent, AoAFs were passaged in SCGM using Trypsin-EDTA solution (Lonza no. CC-5012) for cell detachment. For coculture experiments, THP-1 macrophages were first stimulated with IFN-γ/LPS or IL-4 for 72 hours in complete RPMI 1640 medium in 24-well cell culture inserts (0.4 μm pore, <0.85 × 10⁸ pores/cm², 5 × 10⁴ cells/insert; Thermo Fisher Scientific). The THP-1 medium was replaced with serum-free RPMI 1640 medium, and the inserts were transferred to wells with AoAF (5 × 10⁴ cells/well) in serum-free SCGM. THP-1 macrophages were stimulated with 10 μM PDBu in the presence or absence of 1000 U/ml PEG-catalase (Sigma-Aldrich no. C4963), and the cultures incubated for further 24 hours before lysates were harvested for western blotting.

Antibody against NOX5 was from Abnova (Cat# PAB17793), antibodies against Ki-67 (Cat# 9027), CD31 (Cat# 3528), GAPDH(Cat# 5174), Flag (Cat# 2368), HA (Cat# 2367), c-Abl (Cat# 2862), Src (Cat# 2108) were from CST, antibodies against Pyk2 (Cat# ab226798), α1-ATPase (Cat# ab7671), phosphotyrosine (Cat# ab10321), DUOX2 (Cat# ab97266), Carbonic anhydrase IX (CA IX) (Cat# ab15086) were from Abcam, pSrc (Tyr⁴¹⁹) (Cat# 102-17936) was from Raybiotech, NOX1 (Cat# A8527), NOX2 (Cat# A1636), NOX3 (Cat# A3677), NOX4 (Cat# A11904), DUOX1 (Cat# A8583) were obtained from ABclonal, Src inhibitors-dasatinib (Cat# S1021) and PP2 (Cat# S7008) were purchased from Selleck Chemicals. PEG-catalase (Cat# C4963-2MG), Diphenyleneiodonium (DPI; Cat# D2926-10MG), and N-Acetyl-L-cysteine (NAC; Cat# A9165-5G) were purchased from Sigma-Aldrich.

The assay was done as described [36] (link). Briefly, cells were plated in 12-well dishes 24 h before the experiment. Cells were washed once with warm PBS, followed by incubation with 300 µL of pre-warmed assay buffer [HEPES-buffered Tyrode's solution (no FBS) containing 50 µM Amplex Red (Invitrogen) and 2 U/mL Horse Radish Peroxidase (Sigma)] containing 10 µM LPC 18:1 or PBS (vehicle) for 15 min. Polyethylene glycol catalase (PEG-catalase) 300 U/mL or polyethylene glycol superoxide dismutase (PEG-SOD) 75 U/mL (both Sigma) were added in order to detect catalase-sensitive peroxides and superoxide radicals in the medium. The buffer was then transferred to a black 96 well plate and fluorescence was measured at excitation and emission wavelengths of 540 and 580 nm, respectively. Relative fluorescence units were normalised to cellular protein concentration. Final values represent catalase sensitive H₂O₂ values obtained by subtration of values measured in the presence of catalase from values obtained upon measurements in the absence of catalse.