Fig. 3.

Cardiac glycosides inhibit HIF-1α protein synthesis. (A) Hep3B cells were exposed to vehicle or the HIF-1α inducer cobalt chloride (CoCl₂), desferrioxamine (DFX), or dimethyloxalylglycine (DMOG) in the presence of 100 nM digoxin (+) or vehicle (−) for 24 h and whole cell lysates were subjected to immunoblot assays. (B) Hep3B cells were treated with vehicle or digoxin in the absence (−) or presence (+) of 5 μM MG132. (C) 293 cells were transfected with empty vector (EV) or expression vector encoding FLAG epitope-tagged HIF-1α that was wild-type or double mutant (DM) because of Pro → Ala substitutions at residues 402 and 564 of the protein. The cells were cultured in the presence of 100 nM digoxin (+) or vehicle (−) for 24 h, and cell lysates were subjected to immunoblot assays by using anti-FLAG antibody. (D) Hep3B cells were pretreated for 4 h with vehicle (Veh), 25 nM rapamycin (Rap), 20 μg/ml cycloheximide (Chx), or 100 nM digoxin (Dig), [³⁵S]methionine/cysteine was added for 1 h followed by cell lysis, HIF-1α immunoprecipitation (IP), SDS/PAGE, and autoradiography. Signal intensity was quantified by densitometry.