Batimastat

For in vitro proteolysis studies, 0.5 μg recombinant human PARP-1 (Life Technologies, Carlsbad, CA, USA) was incubated with 0.44 μg affinity-purified, recombinant human ADAMTS-4 (R&D Systems, Minneapolis, MN, USA) in reaction buffer (50 mM Tris, 10 mM CaCl₂, pH 7.5) with a total volume of 25 μl at 37 °C for 1 to 3 hours. The broad-spectrum pan extracellular matrix protease inhibitor batimastat (Sigma-Aldrich; 0.125 mg/ml) was used to inhibit ADAMTS-4 activity. The products of PARP-1 digestion were analysed by western blot.

Dimercaprol (2,3-dimercapto-1-propanol ≥98% iodometric, Cat no: 64046-10 ml), marimastat (≥98% HPLC M2699-5MG), batimastat (SML0041-5MG) and varespladib (≥98% HPLC SML1100-5MG) were purchased from Sigma-Aldrich. Nafamostat mesylate (ab141432 10 mg) was purchased from Abcam and DMPS (2,3-dimercapto-1-propanesulfonic acid sodium salt monohydrate, 95%, Cat no: H56578) from Alfa Aesar. Working stocks (tenfold dilutions from 2 mM to 2 µM) were made using deionized water, with the exception of varespladib and batimastat, for which we used DMSO due to water insolubility.

Cancer cell lines were seeded on plastic dishes or peritoneal tissue and treated with MMP-2/MMP-9 inhibitor III (444251; Merck Chemicals GmbH, Darmstadt, Germany) or batimastat (196440; Sigma–Aldrich Chemie, St. Louis, MO, USA) for the indicated times. Attached GFP-expressing cells were then counted 24 h after seeding.

Chemical and molecular biology reagents were purchased from Fisher Scientific (Pittsburgh, PA) unless otherwise noted. BMS-754807 was purchased from Active Biochemicals Co. Batimastat and ZnCl₂ were purchased from Sigma (St. Louis, MO, United States), MK2206 from ChemieTek (Indianapolis, IN), wortamannin and Rapamycin from Calbiochem (Gibbstown, NJ). The Phospho-Akt antibody was purchased from Cell Signaling Technology (Danvers, MA, United States) and restriction enzymes were purchased from New England BioLabs (Ipswich, MA, United States). Primers, TRIzol, M-MLV reverse transcriptase were purchased from Life Technologies (Carlsbad, CA, United States). Anti-Digoxigenin-AP antibodies was purchased from Roche (Basel, Switzerland). The pT3.Cas 9-UTRglobin vector was a gift from Dr. Yonghua Sun, Institute of Hydrobiology, Chinese Academy of Sciences.

Chemical and molecular biology reagents were purchased from Fisher Scientific (Pittsburgh, PA, USA) unless stated otherwise. BMS-754807 was purchased from JiHe Pharmaceutica (Beijing, China) and NBI-31772 from Tocris Bioscience (Ellisville, MO, USA). Liberase TM, Alizarin Red S, ZnCl₂, and batimastat were purchased from Sigma (St. Louis, MO, USA). MK2206 was purchased from ChemieTek (Indianapolis, IN). Rapamycin was purchased from Calbiochem (Gibbstown, NJ). Antibodies (phospho-Akt, phospho-S6, and phospho-Erk) were purchased from Cell Signaling Technology (Danvers, MA, USA). Restriction enzymes were purchased from New England BioLabs (Ipswich, MA, USA). Primers, cell culture media, antibiotics, TRIzol, M-MLV reverse transcriptase, Alexa Fluor 488 Tyramide SuperBoost Kit, and Tetramethylrhodamine (TMRM) were purchased from Life Technologies (Carlsbad, CA, USA). Anti-Digoxigenin-POD and Anti-Digoxigenin-AP antibodies were purchased from Roche (Basel, Switzerland).

Bafilomycin A1, epoxomicin, bestatin, and EUK134 (chloro[[2,2'-[1,2-ethanediylbis[(nitrilo-κN)methylidyne]]bis[6-methoxyphenolato-κO]]]-manganese) were from Cayman Chemical Company (Ann Arbor, MI, USA). MnTMPyP (Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin) was from ENZO life sciences (Farmingdale, NY, USA). 3-Isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), insulin, hydrogen peroxide, epoxomicin, batimastat, and H₂¹⁸O were from Sigma (St. Louis, MO, USA). Chemical standards used for metabolite identification were from TCI America (Portland, OR, USA) and when unavailable, Sigma.