E4031

Saxitoxin (SRM NIST, Product # 8642a) was received in a solution of 80% acidified water (pH 3.5)/20% ethanol. Nifedipine (Sigma-Aldrich, Product #N7634) was initially dissolved in dimethyl sulfoxide. E4031 (Alomone Lab, Cat. #E-500) was initially dissolved in MilliQ water. The maximum percentage of DMSO in the total media for tissues treated with Nifedipine was 0.02% at 2 μM of drug, which was considered negligible. BaCl₂ (Sigma 202738) was initially dissolved in MilliQ water to 100 mM.
All drugs were dissolved into phenol red free RPMI1640/B27 at 10 times the desired final concentration, and added to tissues by changing 10% of the media to limit potential thermal and mechanical agitation from full media changes. Tissues treated with saxitoxin, Nifedipine, or E4031 were allowed to equilibrate for 15 min at 37 °C before imaging and were tested at the following final concentrations: 10 nM, 100 nM, 500 nM, and 2 μM. Tissues treated with BaCl₂ were allowed to equilibrate for 35 min at 37 °C before imaging and were tested at a final concentration of 0.5 mM BaCl₂.

HT29 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (4.5 g/L glucose) (Corning, Manassas, VA, USA) with 1% GlutaMax (Gibco, Waltham, MA, USA), FET cells in DMEM/F12 (50:50) media (Corning, Manassas, VA, USA), and SW480 cells in DMEM media, supplemented with 10% fetal bovine serum (FBS) (Gemini Bio, West Sacramento, CA, USA) and 1% penicillin (10,000 units/mL)/streptomycin (10,000 µg/mL) (Gibco, Waltham, MA, USA) at 37 °C and 5% CO₂. All antibodies were purchased from Cell Signaling Technologies, Inc (Boston, MA, USA). An anti-KCNH2 antibody was purchased from Santa Cruz (Dallas, Texas, USA) or Alomone Lab (Islrael). Recombinant human TGF-β1 and anti-hTGF-β RII FITC conjugated were purchased from R&D Systems (Minneapolis, MN, USA). E4031 and NS1643 were purchased from Alomone Labs (Jerusalem, Israel). Sanguinarine chloride was purchased from Tocris (Bristol, UK). Cyclosporine A was purchased from LC Laboratories (Woburn, MA, USA).