AT inhibits HRPII-mediated inflammatory responses in HL60 cells. (A) dHL60 cells were treated with HRPII (100 nM) for 5 to 15 minutes followed by the analysis of phosphorylation of NF-κB p65 by western blotting. Densitometric analysis is presented. (B) The same as (A) except that cells were treated simultaneously with HRPII and AT (2.5 µM) followed by the analysis of phosphorylation of NF-κB p65 by western blotting. Densitometric analysis is presented. (C) dHL60 cells on coverslips were treated simultaneously with HRPII (100 nM) and different AT derivatives (AT-WT, AT-N135Q and AT-4Mut (2.5 µM) for 4 hours and then fixed in 4% paraformaldehyde, permeabilized with 0.2% TritonX-100/PBS, and stained with anti-citrullinated H3 (rabbit polyclonal) antibody followed by Alexa Fluor 555-conjugated anti-rabbit antibody. DNA was stained with Sytox Green. (D) dHL60 cells were treated with HRPII (100 nM), TNF-α (10 ng/mL), or PMA (100 nM) for 4 hours followed by analysis of histone (H3) citrullination by western blotting. Densitometric analysis is presented. (E) The same as (D) except that cells were treated with HRPII and different AT derivative (AT-WT, AT-N135Q and AT-4Mut (2.5 µM) and histone (H3) citrullination were analyzed by western blotting. Densitometric analysis is presented.