Penicillin streptomycin g

cDNA encoding hMCT9 tagged with a FLAG epitope (hMCT9-FLAG) was cloned in the mammalian expression vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA). The plasmid was transfected into HEK293 cells using Lipofectamine 2000 (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. To establish a cell line that stably expresses hMCT9-FLAG, HEK293 cells were cultured in the presence of 1 mg/mL geneticin (G418) and the cell clones were then harvested. Expression of hMCT9-FLAG was confirmed by western blotting. Cells stably expressing hMCT9-FLAG were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (Nichirei Biosciences, Tokyo, Japan) and 0.5% penicillin-streptomycin G (Wako) at 37℃ in a 5% CO₂incubator.

cDNA encoding the wild-type hERG protein tagged with the FLAG octapeptide epitope (WT-hERG-FLAG) was cloned in a mammalian expression vector, pcDNA3.1 (+) (Invitrogen, Carlsbad, CA, USA). Individual expression plasmids were transfected into HEK293 cells using Lipofectamine 2000 (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer׳s instructions. To establish the cells stably expressing WT-hERG-FLAG, they were cultured in the presence of 1 mg/mL Geneticin (G418) and cell clones were then harvested. The expression of WT-hERG-FLAG was confirmed by Western blotting.
Cells were cultured in Dulbecco׳s modified Eagle׳s medium (D-MEM; Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (Nichirei Biosciences, Tokyo, Japan) and 0.5% penicillin-streptomycin G (Wako, Osaka, Japan) at 37 °C in a 5% CO₂ incubator in the presence and absence of pilsicainide at 0.03–10 μM.
We also used HEK293 cells stably expressing mutant hERG proteins with a mutation of G601S or R752W, both of which are trafficking-defective. G601S locates in the pore region of hERG channels, while R752W in the intracellular domain, as described elsewhere [15] .