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Fig. <t>5</t> <t>NADPH</t> activation and ROS production were involved in the formation of P. hominis-induced METs. a, Macrophages (7.5 × 105 cells/ml) were pretreated with NADPH oxidase inhibitor (DPI) prior to P. hominis trophozoite stimulation for 30 min. b, ROS production was detected via DCFH-DA. Macrophages were pretreated or untreated with DPI. The macrophages stimulated with zymosan (1 mg/ml) were used as a positive control, and unstimulated macrophages were used as a negative control. c, Macrophages (7.5 × 105 cells/ml) were pretreated with <t>MPO</t> inhibitor (4-ABAH) prior to P. hominis trophozoite stimulation for 30 min. d, DNase I was added to the coincubated medium 15 min before the end of incubation. The dsDNA produced by METs in supernatants was stained with PicoGreen and detected. Bars represent the mean ± SD for the three experiments, ***P < 0.001, ****P < 0.0001
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Fig. <t>5</t> <t>NADPH</t> activation and ROS production were involved in the formation of P. hominis-induced METs. a, Macrophages (7.5 × 105 cells/ml) were pretreated with NADPH oxidase inhibitor (DPI) prior to P. hominis trophozoite stimulation for 30 min. b, ROS production was detected via DCFH-DA. Macrophages were pretreated or untreated with DPI. The macrophages stimulated with zymosan (1 mg/ml) were used as a positive control, and unstimulated macrophages were used as a negative control. c, Macrophages (7.5 × 105 cells/ml) were pretreated with <t>MPO</t> inhibitor (4-ABAH) prior to P. hominis trophozoite stimulation for 30 min. d, DNase I was added to the coincubated medium 15 min before the end of incubation. The dsDNA produced by METs in supernatants was stained with PicoGreen and detected. Bars represent the mean ± SD for the three experiments, ***P < 0.001, ****P < 0.0001
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Fig. <t>5</t> <t>NADPH</t> activation and ROS production were involved in the formation of P. hominis-induced METs. a, Macrophages (7.5 × 105 cells/ml) were pretreated with NADPH oxidase inhibitor (DPI) prior to P. hominis trophozoite stimulation for 30 min. b, ROS production was detected via DCFH-DA. Macrophages were pretreated or untreated with DPI. The macrophages stimulated with zymosan (1 mg/ml) were used as a positive control, and unstimulated macrophages were used as a negative control. c, Macrophages (7.5 × 105 cells/ml) were pretreated with <t>MPO</t> inhibitor (4-ABAH) prior to P. hominis trophozoite stimulation for 30 min. d, DNase I was added to the coincubated medium 15 min before the end of incubation. The dsDNA produced by METs in supernatants was stained with PicoGreen and detected. Bars represent the mean ± SD for the three experiments, ***P < 0.001, ****P < 0.0001
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Fig. 5 NADPH activation and ROS production were involved in the formation of P. hominis-induced METs. a, Macrophages (7.5 × 105 cells/ml) were pretreated with NADPH oxidase inhibitor (DPI) prior to P. hominis trophozoite stimulation for 30 min. b, ROS production was detected via DCFH-DA. Macrophages were pretreated or untreated with DPI. The macrophages stimulated with zymosan (1 mg/ml) were used as a positive control, and unstimulated macrophages were used as a negative control. c, Macrophages (7.5 × 105 cells/ml) were pretreated with MPO inhibitor (4-ABAH) prior to P. hominis trophozoite stimulation for 30 min. d, DNase I was added to the coincubated medium 15 min before the end of incubation. The dsDNA produced by METs in supernatants was stained with PicoGreen and detected. Bars represent the mean ± SD for the three experiments, ***P < 0.001, ****P < 0.0001

Journal: Parasites & vectors

Article Title: Pentatrichomonas hominis induces extracellular traps formation of macrophages via the TLR2/NADPH/PAD4 pathway.

doi: 10.1186/s13071-025-06840-w

Figure Lengend Snippet: Fig. 5 NADPH activation and ROS production were involved in the formation of P. hominis-induced METs. a, Macrophages (7.5 × 105 cells/ml) were pretreated with NADPH oxidase inhibitor (DPI) prior to P. hominis trophozoite stimulation for 30 min. b, ROS production was detected via DCFH-DA. Macrophages were pretreated or untreated with DPI. The macrophages stimulated with zymosan (1 mg/ml) were used as a positive control, and unstimulated macrophages were used as a negative control. c, Macrophages (7.5 × 105 cells/ml) were pretreated with MPO inhibitor (4-ABAH) prior to P. hominis trophozoite stimulation for 30 min. d, DNase I was added to the coincubated medium 15 min before the end of incubation. The dsDNA produced by METs in supernatants was stained with PicoGreen and detected. Bars represent the mean ± SD for the three experiments, ***P < 0.001, ****P < 0.0001

Article Snippet: In parallel experiments, macrophages were pretreated with 20 μM of NADPH oxidase inhibitor (diphenylene iodonium [DPI], Merck, D2926, USA), 100 μM of MPO inhibitor (4-aminobenzoic acid hydrazide [4-ABAH], Selleck, S9874, China), 50 μM of extracellular regulated protein kinase 1/2 (ERK1/2) inhibitor (U0126, Selleck, S1102, China), 10 μM of p38 MAPK inhibitor (SB202190, Selleck, S1077, China), 100 μM of TLR2 inhibitor (TLR2-IN-C29, Selleck, S6597, China), 100 μM of SOCE inhibitor (2-aminoethyl diphenylborinate [2-APB], Selleck, S6657, China), 10 μM of PAD4 inhibitor (GSK484 hydrochloride, MedChemExpress, HY-100514, USA), and 1 μM of cytochalasin D (Cyt D, APExBio, 22144-77- 0, USA) at 37 °C and 5% CO2 for 30 min. DNase I in 90 U/well (Thermo Scientific, EN0523, USA) was used to digest the MET structure for 15 min before the end of coincubation.

Techniques: Activation Assay, Positive Control, Negative Control, Incubation, Produced, Staining