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

Figure 2.

Figure 2

ATP induced PAD4 and CitH3 upregulation in neutrophils and promoted NETosis in a P2X7R-dependent manner. (A–D) Blood PMNs were treated with 50 μM of ATP or BzATP for the indicated duration (A,C) or a range of doses of ATP or BzATP for 4 h (B,D). Levels of CitH3 (A,B) and PAD4 (C,D) were determined by immunoblotting. (E,F) Blood PMNs were pretreated with A438079 (10 or 20 μM) for 20 min before treatment with 100 μM ATP (E) or 50 μM BzATP (F) for 4 h. PAD4 levels were subsequently assessed by immunoblotting. (G,H) Blood PMNs were pretreated with A438079 (10 or 20 μM) (G) or Cl-amidine (10 or 20 μM) (H) for 20 min prior to treatment with 100 μM ATP or BzATP for 4 h. CitH3 levels were determined by immunoblotting. Immunoblots are representative of 2~4 independent experiments. (I–K) Blood PMNs were pretreated with A438079 (10 μM) or Cl-amidine (20 μM) for 20 min prior to treatment with ATP (50, 100, or 200 μM) or with BzATP (50 μM) for 4 h. dsDNA release was visualized by staining with Sytox Green (I) and the amounts of free dsDNA (J) or DNA complexed with neutrophil elastase (NE-DNA) (K) were assessed using Quant-iT PicoGreen dsDNA reagent. Arrows in I indicate the released DNA and scale bars in represent 50 μm. Results are presented as mean ± SEM (n = 3). * p < 0.05, ** p < 0.01 versus the PBS-treated control, ## p < 0.01 versus ATP (100 μM) only-treated cells.