Figure 2.

CQ and HCQ inhibits PAD4 in a dose-dependent manner. (a) PAD4 inhibition was measured through Cayman Chemical PAD4 Inhibitor Screening Assay Kit (Ammonia) per manufacturer’s instructions after treatment with positive control, Cl-Amidine, CQ, or HCQ at the concentrations indicated, demonstrating dose-dependent inhibition of PAD4 for both compounds. (b) Measurement of PAD2 inhibition through Cayman Chemical PAD2 Inhibitor Screening Assay Kit after treatment with Cl-Amidine, CQ, and HCQ showing lack of PAD2 inhibition with CQ and HCQ. (c) NETs were stained with Hoechst for DNA (Blue) and CitH3 (Red, anti-citrullinated histone 3 with an anti-rabbit secondary conjugated to FITC) after stimulation with PAF with and without treatment of hydroxychloroquine or chloroquine and a representative image is shown. CitH3 positive NETs are marked with white arrows and CitH3 negative NETs with green arrows. These findings demonstrated a reduction in CitH3 in HCQ/CQ-treated neutrophils. (d) Quantification of 9 wells from each treatment measuring CitH3 positive NETs.