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Platelet HMGB1 triggers NETosis following PTS in TLR4- and <t>CXCR4-dependent</t> manner. A Schematic illustration of the co-culture protocol for normal PMNs and platelets isolated from PTS animals. B – E Normal PMNs were seeded onto six-well-plate and cultured for 24 h and then co-cultured with platelets isolated from sham-operated or at 1 or 3 h post-PTS animals for 3 h. ( B – E ) PMNs were pre-incubated with HMGB1 A box (100 ng/mL) or A438079 (20 µM) for 30 min before co-culture with platelets. ATP treatment (25 µM) was used as a positive control. Protein expression of CitH3, MPO, and HMGB1 was assessed by immunoblotting ( B , C , E ) and levels of cell-free DNA in serum were measured ( D ). F PMNs were pre-incubated with TLR4-IN-C34 (20 µM), <t>AMD3100</t> (20 µM), or FPS-ZM1 (150 nM) for 30 min before co-culture with platelets. Representative images are shown and quantified data are presented as mean ± SEM (n = 4–6). *p < 0.05, ***p < 0.001 compared to sham group and # p < 0.05, ## p < 0.01, ### p < 0.001, & p < 0.05, &&& p < 0.001, $ p < 0.05 between indicated groups
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Platelet HMGB1 triggers NETosis following PTS in TLR4- and <t>CXCR4-dependent</t> manner. A Schematic illustration of the co-culture protocol for normal PMNs and platelets isolated from PTS animals. B – E Normal PMNs were seeded onto six-well-plate and cultured for 24 h and then co-cultured with platelets isolated from sham-operated or at 1 or 3 h post-PTS animals for 3 h. ( B – E ) PMNs were pre-incubated with HMGB1 A box (100 ng/mL) or A438079 (20 µM) for 30 min before co-culture with platelets. ATP treatment (25 µM) was used as a positive control. Protein expression of CitH3, MPO, and HMGB1 was assessed by immunoblotting ( B , C , E ) and levels of cell-free DNA in serum were measured ( D ). F PMNs were pre-incubated with TLR4-IN-C34 (20 µM), <t>AMD3100</t> (20 µM), or FPS-ZM1 (150 nM) for 30 min before co-culture with platelets. Representative images are shown and quantified data are presented as mean ± SEM (n = 4–6). *p < 0.05, ***p < 0.001 compared to sham group and # p < 0.05, ## p < 0.01, ### p < 0.001, & p < 0.05, &&& p < 0.001, $ p < 0.05 between indicated groups
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Platelet HMGB1 triggers NETosis following PTS in TLR4- and <t>CXCR4-dependent</t> manner. A Schematic illustration of the co-culture protocol for normal PMNs and platelets isolated from PTS animals. B – E Normal PMNs were seeded onto six-well-plate and cultured for 24 h and then co-cultured with platelets isolated from sham-operated or at 1 or 3 h post-PTS animals for 3 h. ( B – E ) PMNs were pre-incubated with HMGB1 A box (100 ng/mL) or A438079 (20 µM) for 30 min before co-culture with platelets. ATP treatment (25 µM) was used as a positive control. Protein expression of CitH3, MPO, and HMGB1 was assessed by immunoblotting ( B , C , E ) and levels of cell-free DNA in serum were measured ( D ). F PMNs were pre-incubated with TLR4-IN-C34 (20 µM), <t>AMD3100</t> (20 µM), or FPS-ZM1 (150 nM) for 30 min before co-culture with platelets. Representative images are shown and quantified data are presented as mean ± SEM (n = 4–6). *p < 0.05, ***p < 0.001 compared to sham group and # p < 0.05, ## p < 0.01, ### p < 0.001, & p < 0.05, &&& p < 0.001, $ p < 0.05 between indicated groups
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Platelet HMGB1 triggers NETosis following PTS in TLR4- and CXCR4-dependent manner. A Schematic illustration of the co-culture protocol for normal PMNs and platelets isolated from PTS animals. B – E Normal PMNs were seeded onto six-well-plate and cultured for 24 h and then co-cultured with platelets isolated from sham-operated or at 1 or 3 h post-PTS animals for 3 h. ( B – E ) PMNs were pre-incubated with HMGB1 A box (100 ng/mL) or A438079 (20 µM) for 30 min before co-culture with platelets. ATP treatment (25 µM) was used as a positive control. Protein expression of CitH3, MPO, and HMGB1 was assessed by immunoblotting ( B , C , E ) and levels of cell-free DNA in serum were measured ( D ). F PMNs were pre-incubated with TLR4-IN-C34 (20 µM), AMD3100 (20 µM), or FPS-ZM1 (150 nM) for 30 min before co-culture with platelets. Representative images are shown and quantified data are presented as mean ± SEM (n = 4–6). *p < 0.05, ***p < 0.001 compared to sham group and # p < 0.05, ## p < 0.01, ### p < 0.001, & p < 0.05, &&& p < 0.001, $ p < 0.05 between indicated groups

Journal: Molecular Medicine

Article Title: Platelet-derived HMGB1 induces NETosis, exacerbating brain damage in the photothrombotic stroke model

doi: 10.1186/s10020-025-01107-7

Figure Lengend Snippet: Platelet HMGB1 triggers NETosis following PTS in TLR4- and CXCR4-dependent manner. A Schematic illustration of the co-culture protocol for normal PMNs and platelets isolated from PTS animals. B – E Normal PMNs were seeded onto six-well-plate and cultured for 24 h and then co-cultured with platelets isolated from sham-operated or at 1 or 3 h post-PTS animals for 3 h. ( B – E ) PMNs were pre-incubated with HMGB1 A box (100 ng/mL) or A438079 (20 µM) for 30 min before co-culture with platelets. ATP treatment (25 µM) was used as a positive control. Protein expression of CitH3, MPO, and HMGB1 was assessed by immunoblotting ( B , C , E ) and levels of cell-free DNA in serum were measured ( D ). F PMNs were pre-incubated with TLR4-IN-C34 (20 µM), AMD3100 (20 µM), or FPS-ZM1 (150 nM) for 30 min before co-culture with platelets. Representative images are shown and quantified data are presented as mean ± SEM (n = 4–6). *p < 0.05, ***p < 0.001 compared to sham group and # p < 0.05, ## p < 0.01, ### p < 0.001, & p < 0.05, &&& p < 0.001, $ p < 0.05 between indicated groups

Article Snippet: For signaling pathway experiments, PMNs were pre-treated with TLR4 antagonist (TLR4-IN-C34, 20 µM, Sigma-Aldrich), C-X-C chemokine receptor type 4 (CXCR4) receptor antagonist (AMD3100, 20 µg/mL, Sigma-Aldrich), and RAGE antagonist (FPS-ZM1, 150 nM, Sigma-Aldrich) and then co-cultured with platelets.

Techniques: Co-Culture Assay, Isolation, Cell Culture, Incubation, Positive Control, Expressing, Western Blot