Cell Invasion Assay with PAD Inhibitors

The cell invasion assay was performed according to previous methods described in detail elsewhere [17 (link),117 (link)]. Briefly, 5 × 10⁵ cells (treated with pan-PAD inhibitor Cl-amidine and the PAD isozyme-specific inhibitors with respective PBS or DMSO control as before) were plated on Matrigel-coated transwell filters (Corning™ BioCoat™ Matrigel™ Invasion Chamber with Corning™ Matrigel Matrix; BD Biosciences, Wokingham, UK) in a chemotactic gradient of 1:10% FBS. Following an incubation time of 16 h, the number of invaded cells was determined using the crystal violet assay (Abcam, U.K.) and the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Abcam, U.K.). The same number of cells was plated and incubated in parallel for 16 h, for assessment of PAD inhibitor-mediated effects on cell proliferation. The CLARIOstar plate reader (BMG Labtech, Aylesbury, UK) was used at 540–590 nm to measure absorbance and normalised according to the control. The experiments were performed in three biological and three technical repeats.