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. 2020 Oct 16;21(20):7668. doi: 10.3390/ijms21207668

Figure 6.

Figure 6

ATP-P2X7R mediates NETosis induction in ischemic brains. (A) Apyrase (0.2 U/g, i.v.) and BzATP (5 mg/kg, i.v.) was administered at 6 h post-MCAO and Trolox (2.5 mg/kg, i.v.) and apocynin (2.5 mg/kg, i.v.) was administered at 9 h post-MCAO. Immunoblotting and immunofluorescence staining was carried out at 12 or 24 h post-MCAO. (B–F) PAD4 and CitH3 levels in blood PMNs or cortical penumbras of ischemic hemispheres were determined at 12 or 24 h post-MCAO, respectively, by immunoblotting with or without apyrase (0.2 U/g, i.v.) treatment at 6 h post-MCAO (B–D), and immunofluorescence staining of brain tissues sections with CitH3 antibody was carried out at 24 h post-MCAO (E,F). (G,I,J) Apocynin (2.5 mg/kg) or Trolox (2.5 mg/kg) was administered 9 h post-MCAO and levels of PAD4, CitH3, and NET formation in blood PMNs purified at 12 h post-MCAO were assessed by immunoblotting (G) or immunofluorescence staining with CitH3 antibody (I,J), respectively. (H) PAD4 and CitH3 levels in cortical penumbra of the ischemic hemisphere were determined at 12 post-MCAO after treating BzATP at 6 h post-MCAO and with or without Trolox (2.5 mg/kg, i.v.) and apocyanin (2.5 mg/kg, i.v.) administration at 9 h post-MCAO. Immunoblots are representative of 2 or 3 independent experiments. Scale bars in (E,I) represent 50 μm. Results are presented as mean ± SEM (n = 3). ** p < 0.01 versus sham control, ## p < 0.01 versus MCAO. Numbers under the blot indicate band intensities.