Nifedipine, amiloride, apamin, 4-AP (4-aminopyridine), and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) were purchased from Merck. AqB013 [47 (link)] was synthesized by Dr G. Flynn (Spacefill Enterprises LLC, Bozeman, MT, USA) and repurified by Dr. N. Proschogo (University of Sydney, NSW, Australia). Xanthurenic acid and caelestine C from the Davis Open Access Natural Product-based Library were provided by R. Davis (Griffith University, QLD Australia). Stock solutions (1000×) of AqB013 (14 mM), nifedipine (25 mM), amiloride (10 mM), CNQX (30 mM), xanthurenic acid (1 mM), and caelestine C (1 mM) were prepared in DMSO. DMSO alone at 0.1% served as the vehicle control for DMSO-solubilized compounds. Stock solutions (1000×) of apamin (10 mM) and 4-aminopyridine (250 mM) were prepared in water; for these, media without DMSO served as the control comparison. The tested concentrations of AqB013, nifedipine, amiloride, CNQX, apamin, and 4-aminopyridine were selected based on parameters established in the literature [48 (link),49 (link),50 (link),51 (link),52 (link)]. For the novel compounds, xanthurenic acid and caelestine C, dose-response experiments were conducted to determine the concentrations to be used for the cellular motility assays.
For testing in transwell invasion assays, all pharmacological agents were diluted at 1 µL/mL in final media. For testing in spheroid spreading assays, all pharmacological agents were diluted at 2 µL/mL in final media and DMSO alone at 0.2% served as the vehicle control for DMSO-solubilized compounds.
For testing in transwell invasion assays, all pharmacological agents were diluted at 1 µL/mL in final media. For testing in spheroid spreading assays, all pharmacological agents were diluted at 2 µL/mL in final media and DMSO alone at 0.2% served as the vehicle control for DMSO-solubilized compounds.
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