Eplerenone

NHEKs (Lonza, Basel, Switzerland) within two or three passages were cultured in KBM-Gold medium supplemented with a KGM-Gold Bullet Kit (Lonza) at 37 °C and 5% CO₂. Hydrocortisone, one of the components of the KGM-Gold Bullet Kit, was not added to the medium for the assay. NHEKs were treated with cortisol (10 μM) in the presence or absence of 7,3’,4’-THIF (Indofine Chemical Co., Inc., Hillsborough, NJ, USA), mifepristone (Tocris, Bristol, UK), or eplerenone (Tocris), and harvested 4 days later for further analysis.

HEK293 cells were grown in DMEM (low-glucose, Gibco) supplemented with 10% FBS (Wisent Bioproducts), 100 units/ml penicillin, and 100 μg/ml streptomycin (Gibco). 400 μg/ml zeocin (Invitrogen) and 5 μg/ml blasticidin (Invitrogen) were further added to maintain stable expression of TRPM7 constructs. Cells were cultured at 37 °C in 5% CO₂. Cell medium was changed every 3–4 days, and cells were passaged every 4–5 days via trypsinization.
HEK293 cells were stably transfected with HA-tagged WT hTRPM7 or phosphotransferase-inactive K1648R hTRPM7 in pcDNA4/TO vectors (38 (link), 66 (link)). This expression system has been used in prior electrophysiological studies of TRPM7 (33 (link), 35 (link), 40 (link), 80 (link)). TRPM7 expression was induced by the addition of 1 μg/ml tetracycline (Sigma) 18 h prior to experimentation, as described previously (38 (link), 66 (link)). In experiments examining aldosterone, cells were concurrently induced with tetracycline (18 h) and treated (18 h) with 100 nm aldosterone (Sigma) (0.004% ethanol vehicle) unless indicated otherwise. In experiments utilizing antagonists, cells were also treated (18 h) with 360 nm eplerenone (0.1% DMSO vehicle) (Tocris) or 6 μm GSK-650394 (0.1% DMSO vehicle) (Tocris). Cell induction and treatments were performed on 60–90% confluent HEK293 cells grown in 60- or 100 mm-diameter Petri dishes.