Diphenyleneiodonium chloride dpi

PMs were isolated from WT or Nlrp3^−/− mice and infected with N. caninum at a multiplicity of infection (MOI) of 3:1, 2:1 or 1:1 (parasite: cell) in R-1% medium for 2, 4 or 8 h, then PMs were washed twice with PBS to remove non-adhered parasites. In the experimental group, cells were pre-treated with the ROS inhibitor NAC for 1 h (5 mM; Selleck, Shanghai, China), or the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI, 10 μM; Selleck) for 2 h [30 (link)]. Additionally, at 2, 4 or 8 h post-infection (pi), the medium was changed to fresh medium with or without the ROS inducer pyrogallol (PG; Selleck). PMs cultured with the equivalent volume of R-1% medium was used as a negative control. Adenosine triphosphate (ATP) is a NLRP3 inflammasome inducer [31 (link)], so PMs in positive control group were pre-treated with N. caninum and then stimulated with 5 mM ATP (Sigma-Aldrich, Shanghai, China) for 30 min. At 5, 24 or 36 h post-infection, cells and supernatants were collected for subsequent experiments, as described below.

(+)-Borneol was obtained from the Simcere Pharmaceutical Group. Polymorphprep was obtained from Axis–Shield. RPMI-1640 was obtained from Gibco. PMA and the NETosis assay kit were obtained from Cayman Chemical. HEPES buffer, enhanced cell counting kit-8 (CCK-8), and ROS assay kit were obtained from Beyotime Biotechnology. The Quant-iT PicoGreen dsDNA Assay Kit was obtained from Invitrogen. SYTOX Green nucleic acid stain and Calcein Blue AM were obtained from Maokang Biotechnology. Diphenyleneiodonium chloride (DPI) and Go6976 were obtained from Selleck. C29 and TAK-242 were obtained from MedChemExpress.
(+)-Borneol was dissolved in DMSO (80 mg/ml) and then diluted with RPMI-1640 to different concentrations (1.56, 6.25, 25, 100, and 400 μM). PMA (1 mg/ml in DMSO) was diluted with RPMI-1640 to 100 nM. The vehicle group had no (+)-borneol or inhibitor but the same PMA and DMSO level as the 400-μM (+)-borneol group. In the control group, neutrophils were treated without PMA.

Cell lines were cultured under the following conditions: PC3, C42B, RM1, PAIII- DMEM (Hyclone SH30243.01), 10% FBS (PEAK PS-FB1), 1% penicillin/streptomycin (P/S) (Gibco 15140-122) and LNCaP- RPMI (Hyclone SH30027.02), 10% FBS, 1% P/S. ROS inhibitors: Apocynin 100uM (Millipore 498-02-2), gp91 ds-tat 10uM (Anaspec AS-63818), sgp91 10uM (Anaspec 63821), phorbol 12-myristate 13-acetate (PMA) 100 nM (Caymen 10008014), Rotenone 5uM (Sigma 13995), N-acetyl-l-cysteine (NAC) 5 mM (Sigma 1009005) and Diphenyleneiodonium chloride (DPI) (Selleckchem S8639) and 100 μM Butathione Sulfoximine (BSO) (Caymen 14484). All drugs were used according to manufacturers’ instructions. Prostate cancer conditioned media (CM) was collected as previously described in serum free RPMI [12 (link)].