Marimastat

To determine the enzymatic specificity of FIB One, the probe was incubated at a concentration of 1 μM (unless otherwise stated) with the recombinant MMPs (Enzo Life Sciences) (active domains of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, and MMP-13, all at 30 nM), thrombin (Sigma Aldrich) (5 U/mL), factor Xa (Sigma Aldrich) (500 nM), plasmin (Sigma Aldrich) (30 nM), and human neutrophil elastase (30 nM). Where appropriate, enzymes were incubated with specific inhibitors for 30 min at 37°C prior to addition of FIB One. The pan-MMP inhibitor marimastat (Tocris Biosciences) was used at 20 μM, and the MMP inhibitor AZD1236 was used at 0-14 μM. Enzyme free reactions served as a control. Enzymatic reactions were performed in MMP buffer (10 mM CaCl₂, 6.1 g Tris-HCl, 8.6 g NaCl per litre, pH 7.5) in a final volume of 20 μL. Reactions were performed in duplicate in 384-well plates (Life Technologies) with optically clear plate seals (Thermo Scientific) and repeated thrice (independently). Fluorescence signal was measured for up to 60 min, ex/em 485/528 nm using microplate reader (BioTek Synergy H1 multimode reader). Data were normalised to buffer alone and are presented as fold-change in signal (relative fluorescent units, RFU) compared to enzyme-free controls. Data were plotted using Prism 7 (GraphPad Software Inc., La Jolla, CA, USA).

The EGFR kinase domain inhibitors were purchased from Santa Cruz (Gefitinib), Selleckchem (PD153035), Tocris Bioscience (AG1478) and Sigma (GW2974). Marimastat, GI-253023X, glibenclamide and Bapta-AM were purchased from Tocris Bioscience. Inhibitors were reconstituted in DMSO and aliquoted for appropriate freezer temperature storage. TR146 cells were incubated with inhibitors for 1 h prior to C. albicans infection, candidalysin exposure or mock treatment. Phospho-EGFR Tyr1068 (#3777), phospho-EGFR Tyr845 (#6963S), c-Fos (#2250S) and phospho-MKP1 (#2857S) antibodies were purchased from Cell Signalling Technology. Mouse anti-human α-actin antibody was purchased from Millipore (UK) (#MAB1501), goat anti-mouse (#115-035-062) and anti-rabbit (#111-035-144) horseradish peroxidase (HRP)-conjugated antibodies were purchased from Jackson Immunologicals (Stratech Scientific, UK). Fluorescent ErbB receptor affibodies were a kind gift from the Science and Technologies Facilities Council for use with confocal imaging work. APC anti-human EGFR antibody was purchased from BioLegend (# 352905) for use with Imagestream analyses. Biologically active EREG and EPG were purchased from Peprotech and used at 10, 25 and 50 ng/mL either individually or together at the same concentration.

Annexin V-AF568 and Hoechst 33342 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). O-phospho-L-serine (OPS) and Phorbol 12-myristate-13-acetate (PMA) were obtained from Sigma Aldrich (St. Louis, MO, USA). Ionomycin (IO) was purchased from Merck Millipore (Darmstadt, Germany). Hydroxamate-based ADAM17/ADAM10 inhibitor GW280264X [30 (link)] was purchased from Aeobious (Gloucester, MA, USA). Marimastat and ADAM10 inhibitor GI254023X [31 (link)] were purchased from Tocris Bioscience (Bristol, UK). Additional antibodies used: ANO1 (Bio-Techne, Abingdon, UK), ANO4 (Aviva Systems Biology, San Diego, CA, USA), ANO5 (Abcam, Cambridge, UK), ANO6 and ANO7 (OriGene, Rockville, MD, USA), ANO9 and ANO10 (LSBio, Seattle, WA, USA), and GAPDH (Novus Biologicals, Littleton, CO, USA). Secondary antibodies for automated Western were obtained from Protein Simple (San Jose, CA, USA) or Novus Biologicals (Novus Biologicals, Littleton, CO, USA).