Dronedarone

Cells were plated at a density of 1 × 10⁴ per well in 96-well plates and incubated for 12–16 h to allow cells to adhere. After 24 h, 0.5 mg/mL 3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma) was added, and the cells were incubated for 4 h. After incubation, 100 μl of 10% SDS/0.01N HCl solution were added to each well, followed by shaking for 5 min. Then, the absorbance was measured at 570 nm. Results are representative of three independent experiments, each done in quintuplicate. Since in vitro studies have reported that Amiodarone may be cytotoxic, we performed an MTT assay to determine the least cytotoxic concentration of Amiodarone. Amiodarone was tested at concentrations ranging from 5 to 30 μM. 15 μM was the highest concentration at which all three cell lines tested maintained viability (Supplementary Figure S1). Therefore, 24 hr treatment with 15 μM Amiodarone was used throughout the subsequent cell based, in vitro studies experiments. For other drug treatments, 20 or 50 mg/ml EGF (Peprotech) or (2′Z, 3′E)-6-bromoindirubin -3′-oxime (BIO; 1 μM; EMD Biosciences) were treated with cells for 1 hr, wash out by PBS buffer several times and then for further experiments. AG-1478 (30 uM; Merck), Cetuximab (75 μg/ml; 32; Merck) and Dronedarone (5 μM; Sigma) were treated with cells for 24 hr.

For sodium currents (I_Na), whole-cell patch-clamp recordings were obtained as described previously.⁷ (link)^,8 (link) I_Na was elicited in 100-ms steps to –30 mV from holding potentials of –100 to –70 mV. Currents were measured before and after propafenone (300 nM or 1 μM; Sigma-Aldrich, Gillingham, UK), dronedarone (5 and 10 μM; Sigma-Aldrich, Gillingham, UK), and flecainide (1 μM; Sigma-Aldrich, Gillingham, UK). These concentrations are consistent with those previously reported in the literature.⁷ (link)^,9 (link)^,10 (link) Cells were paced at 1 Hz while the drugs were introduced to the perfusate.
Action potentials (APs) were recorded at 36°–37°C in the whole-cell current-clamp configuration and triggered by 2-ms current injections (1.5 nA). AP trains were stimulated at 1 Hz. APs were recorded at RMPs from –90 to –65 mV by varying the background current injection. Action potential amplitude (APA) and peak AP phase 0 upstroke velocity (dV·dt^-1) were analyzed using modified algorithms from ElectroMap.¹¹ (link)