Veratrine

The following reagents were obtained from Sigma-Aldrich (Gillingham, UK); amiloride, atropine, bumetanide, capsaicin, carbachol, forskolin, veratrine, veratridine.
Tetrodotoxin came from Tocris Bioscience and 4-chlorobenzo(F)isoquinoline from Ubichem plc. Note veratrine is predominantly veratridine together with other similarly acting alkaloids of natural origin.

Solvents for extraction and LC–MS (acetone, n-hexane/s, dichloromethane, acetonitrile, methanol and water) were of HPLC or Optima LC–MS grade and from Fisher Scientific (Hampton, NH, USA). Formic and acetic acid (both pro analysis grade), sodium borohydride (≥98.5%), sodium borodeuteride (98 atom-% D), sodium periodate (≥99.8%) and methanol-d₄, (≥99.8 atom-% D) were from Sigma–Aldrich (St. Louis, MO, USA). All cell culture and assay reagents (DMSO, ouabain, veratrine, phosphate-buffered saline, trypsin, and methylthiazolyldiphenyltetrazolium bromide (MTT), supplements (fetal bovine serum; l-glutamine; sodium pyruvate, penicillin–streptomycin), and Roswell Park Memorial Institute (RPMI) culture medium were of the highest grade available and sourced from Sigma–Aldrich (St. Louis, MO, USA) and Fisher Scientific (Hampton, NH, USA). CTX3C was from Wako Chemicals USA (Richmond, VA, USA). Supplemented RPMI medium contained heat-inactivated fetal bovine serum (5% by volume), sodium pyruvate (1 mM), l-glutamine (2 mM), penicillin (50 units/mL), and streptomycin (50 ng/mL).

Cells were purchased from American Type Culture Collection (VA, USA) and they were grown in the recommended medium. Veratridine, Veratrine, etoposide, 5-Fluorouracil and staurosporine were purchased from Sigma (MO, USA). Because we used DMSO as a solvent for VTD at different concentration (10–300 μM), we examined different concentrations of DMSO (0.02%–0.3%) corresponding to VTD used (10–300 μM) in HCT-116 cells. As the results show in Panel D of Supplementary Figure S1, concentrations of DMSO as a vehicle had no cytotoxic effect on HCT-116 colon cancer measured by MTT assay at 24, 48, and 72 hours. This set of data further confirmed the cytotoxic effect observed with VTD is purely related to the anti-cancer function of VTD and not DMSO.